U.S. patent application number 11/874187 was filed with the patent office on 2008-11-20 for methods and compositions for assessment of pulmonary function and disorders.
This patent application is currently assigned to SYNERGENZ BIOSCIENCE LIMITED. Invention is credited to Robert Peter YOUNG.
Application Number | 20080286776 11/874187 |
Document ID | / |
Family ID | 39314479 |
Filed Date | 2008-11-20 |
United States Patent
Application |
20080286776 |
Kind Code |
A1 |
YOUNG; Robert Peter |
November 20, 2008 |
Methods and Compositions for Assessment of Pulmonary Function and
Disorders
Abstract
The present invention provides methods for the assessment of
risk of developing lung cancer in smokers and non-smokers using
analysis of genetic polymorphisms. The present invention also
relates to the use of genetic polymorphisms in assessing a
subject's risk of developing lung cancer, and the suitability of a
subject for an intervention in respect of lung cancer. Nucleotide
probes and primers, kits, and microarrays suitable for such
assessment are also provided.
Inventors: |
YOUNG; Robert Peter;
(Auckland, NZ) |
Correspondence
Address: |
SONNENSCHEIN NATH & ROSENTHAL LLP
P.O. BOX 061080, WACKER DRIVE STATION, SEARS TOWER
CHICAGO
IL
60606-1080
US
|
Assignee: |
SYNERGENZ BIOSCIENCE
LIMITED
Tortola
VG
|
Family ID: |
39314479 |
Appl. No.: |
11/874187 |
Filed: |
October 17, 2007 |
Current U.S.
Class: |
435/6.14 ;
506/16; 506/18; 536/23.1; 536/24.31; 536/24.33 |
Current CPC
Class: |
C12Q 2600/158 20130101;
C12Q 2600/172 20130101; C12Q 1/6883 20130101; C12Q 1/6886 20130101;
A61P 35/00 20180101; C12Q 2600/156 20130101; C12Q 2600/106
20130101 |
Class at
Publication: |
435/6 ;
536/24.31; 536/24.33; 506/16; 536/23.1; 506/18 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C40B 40/06 20060101
C40B040/06; C40B 40/10 20060101 C40B040/10 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 2006 |
NZ |
NZ 550643 |
Nov 22, 2006 |
NZ |
NZ 551534 |
Dec 7, 2006 |
NZ |
NZ 551883 |
Apr 23, 2007 |
NZ |
NZ 554707 |
Jul 31, 2007 |
NZ |
NZ 560262 |
Jul 31, 2007 |
NZ |
NZ 560263 |
Claims
1. A method of determining a subject's risk of developing lung
cancer comprising analysing a sample from said subject for the
presence or absence of one or more polymorphisms selected from the
group consisting of: Ser307Ser G/T (rsIO56503) in the X-ray repair
complementing defective repair in Chinese hamster cells 4 gene, A/T
c74delA in the gene encoding cytochrome P450 polypeptide CYP3A43;
A/C (rs2279115) in the gene encoding B-cell CLL/lymphoma 2; A/G at
+3100 in the 3' UTR (rs2317676) of the gene encoding Integrin beta
3; -3714 G/T (rs6413429) in the gene encoding Dopamine transporter
1; A/G (rs 1139417) in the gene encoding Tumor necrosis factor
receptor 1; C/Del (rs1799732) in the gene encoding Dopamine
receptor D2; C/T (rs763110) in the gene encoding Fas ligand; C/T
(rs5743836) in the gene encoding Toll-like receptor 9; or one or
more polymorphisms in linkage disequilibrium with one or more of
said polymorphisms, wherein the presence or absence of said
polymorphism is indicative of the subject's risk of developing lung
cancer.
2. A method according to claim 1 wherein the lung cancer is
selected from the group consisting of non-small cell lung cancer
including adenocarcinoma and squamous cell carcinoma, small cell
lung cancer, carcinoid tumor, lymphoma, or metastatic cancer.
3. A method according to claim 1 wherein the method comprises
analysing said sample for the presence or absence of one or more
further polymorphisms selected from the group consisting of: R19W
A/G (rs11015703) in the gene encoding Cerberus 1 (Cer 1); K3326X
A/T (rs11571833) in the breast cancer 2 early onset gene (BRCA2);
V433M A/G (rs2306022) in the gene encoding Integrin alpha-1; E375G
T/C (rs7214723) in the gene encoding Calcium/calmodulin-dependent
protein kinase kinase 1 (CAMKK1); -81 C/T (rs 2273953) in the 5'
UTR of the gene encoding Tumor protein P73 (P73); or one or more
polymorphisms which are in linkage disequilibrium with one or more
of these polymorphisms.
4. A method according to any one of claims 1 to 3 wherein the
presence of one or more of the polymorphisms selected from the
group consisting of: the E375G T/C TT genotype in the gene encoding
CAMKK1; the -81 C/T (rs 2273953) CC genotype the gene encoding P73;
the A/C (rs2279115) AA genotype in the gene encoding BCL2; the
+3100 A/G (rs2317676) AG or GG genotype in the gene encoding ITGB3;
the C/Del (rs1799732) CDel or DelDel genotype in the gene encoding
DRD2; or the C/T (rs763110) TT genotype in the gene encoding Fas
ligand; is indicative of a reduced risk of developing lung
cancer.
5. A method according to any one of claims 1 to 4 wherein the
presence of one or more of the polymorphisms selected from the
group consisting of: the Ser307Ser G/T GG or GT genotype in the
gene encoding XRCC4; the R19W A/G AA or GG genotype in the gene
encoding Cer 1; the Ser307Ser G/T GG or GT genotype in the XRCC4
gene; the K3326X A/T AT or TT genotype in the BRCA2 gene; the V433M
A/G AA genotype in the gene encoding Integrin alpha-1; the A/T
c74delA AT or TT genotype in the gene encoding CYP3A43; the -3714
G/T (rs6413429) GT or TT genotype in the gene encoding DAT 1; the
A/G (rs 139417) AA genotype in the gene encoding TNFR1; or the C/T
(rs5743836) CC genotype in the gene encoding TLR9; is indicative of
an increased risk of developing lung cancer.
6. A method according to any one of claims 1 to 3 wherein the
method comprises analysing each of the polymorphisms of the group
consisting of: -133 G/C (rs360721) in the promoter of the gene
encoding Interleukin-18; -251 A/T (rs4073) in the gene encoding
Interleukin-8; Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in the gene
encoding .alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the
gene encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; or one or more
polymorphisms in linkage disequilibrium with any one or more of
these polymorphisms.
7. A method according to any one of claims 1 to 3 wherein the
method comprises analysing each of the polymorphisms of the group
consisting of: -133 G/C (rs360721) in the promoter of the gene
encoding Interleukin-18; -251 A/T (rs4073) in the gene encoding
Interleukin-8; Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in the gene
encoding .alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the
gene encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115) in the
gene encoding BCL2; or one or more polymorphisms in linkage
disequilibrium with any one or more of these polymorphisms.
8. A method according to any one of claims 1 to 3 wherein the
method comprises analysing each of the polymorphisms of the group
consisting of: -133 G/C (rs360721) in the promoter of the gene
encoding Interleukin-18; -251 A/T (rs4073) in the gene encoding
Interleukin-8; Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in the gene
encoding .alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the
gene encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115) in the
gene encoding BCL2; V433M A/G (rs2306022) in the gene encoding
ITGA11; or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
9. A method according to any one of claims 1 to 3 wherein the
method comprises analysing each of the polymorphisms of the group
consisting of: Rsa 1 C/T (rs2031920) in the gene encoding CYP 2E1;
-133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18; -251 A/T (rs4073) in the gene encoding
Interleukin-8; -511 A/G (rs 16944) in the gene encoding Interleukin
1B; V433M A/G (rs2306022) in the gene encoding ITGA11; Arg 197 Gln
A/G (rs 1799930) in the gene encoding N-acetylcysteine transferase
2; Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; R19W A/G (rs 10115703) in the gene
encoding Cerberus 1; -3714 G/T (rs6413429) in the gene encoding DAT
1; A/G (rs1139417) in the gene encoding TNFR1; C/T (rs5743836) in
the gene encoding TLR9; -81 C/T (rs 2273953) in the 5' UTR of the
gene encoding P73; Arg 312 Gln (rs 1799895) in the gene encoding
SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding
ITGB3; C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115)
in the gene encoding BCL2; -751 G/T (rs 13181) in the promoter of
the gene encoding XPD; Phe 257 Ser C/T (rs3087386) in the gene
encoding REV1; C/T (rs763110) in the gene encoding FasL; or one or
more polymorphisms in linkage disequilibrium with any one or more
of these polymorphisms.
10. A method of assessing a subject's risk of developing lung
cancer said method comprising the steps: (i) determining the
presence or absence of at least one protective polymorphism
associated with a reduced risk of developing lung cancer; and (ii)
in the absence of at least one protective polymorphisms,
determining the presence or absence of at least one susceptibility
polymorphism associated with an increased risk of developing lung
cancer; wherein the presence of one or more of said protective
polymorphisms is indicative of a reduced risk of developing lung
cancer, and the absence of at least one protective polymorphism in
combination with the presence of at least one susceptibility
polymorphism is indicative of an increased risk of developing lung
cancer.
11. A method according to claim 10 wherein said at least one
protective polymorphism is selected from the group consisting of:
the E375G T/C TT genotype in the gene encoding CAMKK1; the -81 C/T
(rs 2273953) CC genotype the gene encoding P73; the A/C (rs2279115)
AA genotype in the gene encoding BCL2; the +3100 A/G (rs2317676) AG
or GG genotype in the gene encoding ITGB3; the C/Del (rs1799732)
CDel or DelDel genotype in the gene encoding DRD2; or the C/T
(rs763110) TT genotype in the gene encoding Fas ligand.
12. A method according to claim 10 or 11 wherein said at least one
susceptibility polymorphism is a genotype selected from the group
consisting of: the Ser307Ser G/T GG or GT genotype in the gene
encoding XRCC4; the R19W A/G AA or GG genotype in the gene encoding
Cer 1; the Ser307Ser G/T GG or GT genotype in the XRCC4 gene; the
K3326X A/T AT or TT genotype in the BRCA2 gene; the V433M A/G AA
genotype in the gene encoding Integrin alpha-1; the A/T c74delA AT
or TT genotype in the gene encoding CYP3A43; the -3714 G/T
(rs6413429) GT or TT genotype in the gene encoding DAT 1; the A/G
(rs1139417) AA genotype in the gene encoding TNFR1; or the C/T
(rs5743836) CC genotype in the gene encoding TLR9.
13. A method according to any one of claims 10 to 12 wherein the
presence of two or more protective polymorphims irrespective of the
presence of one or more susceptibility polymorphisms is indicative
of reduced risk of developing lung cancer.
14. A method according to any one of claims 10 to 12 wherein in the
absence of a protective polymorphism the presence of one or more
susceptibility polymorphisms is indicative of an increased risk of
developing lung cancer.
15. A method according to any one of claims 10 to 12 wherein the
presence of two or more susceptibility polymorphisms is indicative
of an increased risk of developing lung cancer.
16. A method of determining a subject's risk of developing lung
cancer, comprising analysing a sample from said subject for the
presence of two or more polymorphisms selected from the group
consisting of: the Ser307Ser G/T polymorphism in the X-ray repair
complementing defective repair in Chinese hamster cells 4 gene
(XRCC4); R19W A/G in the gene encoding Cerberus 1 (Cer 1); K3326X
A/T in the breast cancer 2 early onset gene (BRCA2); V433M A/G in
the gene encoding Integrin alpha-1; E375G T/C in the gene encoding
Calcium/calmodulin-dependent protein kinase kinase 1 (CAMKK1); A/T
c74delA in the gene encoding cytochrome P450 polypeptide CYP3A43;
A/C (rs2279115) in the gene encoding B-cell CLL/lymphoma 2; A/G at
+3100 in the 3' UTR (rs2317676) of the gene encoding Integrin beta
3; -3714 G/T (rs6413429) in the gene encoding Dopamine transporter
1; A/G (rs 1139417) in the gene encoding Tumor necrosis factor
receptor 1; C/Del (rs1799732) in the gene encoding Dopamine
receptor D2; C/T (rs763110) in the gene encoding Fas ligand; C/T
(rs5743836) in the gene encoding Toll-like receptor 9; -81 C/T (rs
2273953) in the 5' UTR of the gene encoding Tumor protein P73
(P73); or one or more polymorphisms which are in linkage
disequilibrium with any one or more of these polymorphisms.
17. A method according to any one of claims 1 to 16 wherein said
method comprises the analysis of one or more epidemiological risk
factors.
18. A method of determining a subject's risk of developing lung
cancer, said method comprising the steps: (i) obtaining the result
of one or more genetic tests of a sample from said subject; and
(ii) analysing the result for the presence or absence of one or
more polymorphisms selected from the group consisting of: Ser307Ser
G/T in the X-ray repair complementing defective repair in Chinese
hamster cells 4 gene (XRCC4); A/T c74delA in the gene encoding
cytochrome P450 polypeptide CYP3A43; A/C (rs2279115) in the gene
encoding B-cell CLL/lymphoma 2; A/G at +3100 in the 3' UTR
(rs2317676) of the gene encoding Integrin beta 3; -3714 G/T
(rs6413429) in the gene encoding Dopamine transporter 1; A/G
(rs1139417) in the gene encoding Tumor necrosis factor receptor 1;
C/Del (rs1799732) in the gene encoding Dopamine receptor D2; C/T
(rs763110) in the gene encoding Fas ligand; C/T (rs5743836) in the
gene encoding Toll-like receptor 9; or one or more polymorphisms
which are in linkage disequilibrium with one or more of these
polymorphisms; wherein a result indicating the presence or absence
of one or more of said polymorphisms is indicative of the subject's
risk of developing lung cancer.
19. A method according to claim 18 wherein a result indicating the
presence of one or more of the Ser307Ser G/T TT genotype in the
gene encoding XRCC4; the -81 C/T (rs 2273953) CC genotype the gene
encoding P73; the A/C (rs2279115) AA genotype in the gene encoding
BCL2; the +3100 A/G (rs2317676) AG or GG genotype in the gene
encoding ITGB3; the C/Del (rs1799732) CDel or DelDel genotype in
the gene encoding DRD2; or the C/T (rs763110) TT genotype in the
gene encoding Fas ligand; is indicative of a reduced risk of
developing lung cancer.
20. A method according to claim 18 wherein a result indicating the
presence of one or more of: the Ser307Ser G/T GG or GT genotype in
the gene encoding XRCC4; the A/T c74delA AT or TT genotype in the
gene encoding CYP3A43; the -3714 G/T (rs6413429) GT or TT genotype
in the gene encoding DAT 1; the A/G (rs1139417) AA genotype in the
gene encoding TNFR1; or the C/T (rs5743836) CC genotype in the gene
encoding TLR9; is indicative of an increased risk of developing
lung cancer.
21. The method according to any one of claims 18 to 20 additionally
comprising analysing the result for the presence or absence of one
or more further polymorphisms selected from the group consisting
of: R19W A/G in the gene encoding Cerberus 1 (Cer 1); K3326X A/T in
the breast cancer 2 early onset gene (BRCA2); V433M A/G in the gene
encoding Integrin alpha-1; E375G T/C in the gene encoding
Calcium/calmodulin-dependent protein kinase kinase 1 (CAMKK1); -81
C/T (rs 2273953) in the 5' UTR of the gene encoding Tumor protein
P73 (P73); or one or more polymorphisms which are in linkage
disequilibrium with any or more of these polymorphisms.
22. A method according to any one of claims 18 to 21 comprising
analysing the result for the presence or absence of each of the
polymorphisms selected from the group consisting of: -133 G/C
(rs360721) in the promoter of the gene encoding Interleukin-18;
-251 A/T (rs4073) in the gene encoding Interleukin-8; Arg 197 Gln
(rs 1799930) in the gene encoding N-acetylcysteine transferase 2;
Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the gene
encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; or one or more
polymorphisms in linkage disequilibrium with any one or more of
these polymorphisms.
23. A method according to any one of claims 18 to 21 comprising
analysing the result for the presence or absence of each of the
polymorphisms selected from the group consisting of: -133 G/C
(rs360721) in the promoter of the gene encoding Interleukin-18;
-251 A/T (rs4073) in the gene encoding Interleukin-8; Arg 197 Gln
(rs 1799930) in the gene encoding N-acetylcysteine transferase 2;
Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the gene
encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs 1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115) in the
gene encoding BCL2; or one or more polymorphisms in linkage
disequilibrium with any one or more of these polymorphisms.
24. A method according to any one of claims 18 to 21 comprising
analysing the result for the presence or absence of each of the
polymorphisms selected from the group consisting of: -133 G/C
(rs360721) in the promoter of the gene encoding Interleukin-18;
-251 A/T (rs4073) in the gene encoding Interleukin-8; Arg 197 Gln
(rs 1799930) in the gene encoding N-acetylcysteine transferase 2;
Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the gene
encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs 1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115) in the
gene encoding BCL2; V433M A/G (rs2306022) in the gene encoding
ITGA11; or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
25. A method according to any one of claims 18 to 21 comprising
analysing the result for the presence or absence of each of the
polymorphisms selected from the group consisting of: Rsa 1 C/T
(rs2031920) in the gene encoding CYP 2E1; G/C (rs360721) in the
promoter of the gene encoding lnterleukin-18;-251 A/T (rs4073) in
the gene encoding Interleukin-8; -511 A/G (rs 16944) in the gene
encoding Interleukin 1B; V433M A/G (rs2306022) in the gene encoding
ITGA11; Arg 197 Gln A/G (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in the gene
encoding .alpha.1-antichymotrypsin; R19W A/G (rs 10115703) in the
gene encoding Cerberus 1; -3714 G/T (rs6413429) in the gene
encoding DAT 1; A/G (rs1139417) in the gene encoding TNFR1; C/T
(rs5743836) in the gene encoding TLR9; -81 C/T (rs 2273953) in the
5' UTR of the gene encoding P73; Arg 312 Gln (rs 1799895) in the
gene encoding SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the
gene encoding ITGB3; C/Del (rs1799732) in the gene encoding DRD2;
A/C (rs2279115) in the gene encoding BCL2; -751 G/T (rs 13181) in
the promoter of the gene encoding XPD; Phe 257 Ser C/T (rs3087386)
in the gene encoding REV1; C/T (rs763110) in the gene encoding
FasL;
26. or one or more polymorphisms in linkage disequilibrium with any
one or more of these polymorphisms. One or more nucleotide probes
and/or primers for use in the method of any one of claims 1 to 21
wherein the one or more nucleotide probes and/or primers span, or
are able to be used to span, the polymorphic regions of the genes
in which the polymorphism to be analysed is present.
27. One or more nucleotide probes and/or primers as claimed in
claim 26 comprising the sequence of any one of SEQ.ID.NO. 1 to
SEQ.ID.NO. 72.
28. A nucleic acid microarray which comprises a substrate
presenting nucleic acid sequences capable of hybridizing to nucleic
acid sequences which encode one or more of the polymorphisms
selected from the group defined in claim 1 or sequences
complimentary thereto.
29. The use of one or more polymorphisms selected from the group
consisting of: Ser307Ser G/T polymorphism in the X-ray repair
complementing defective repair in Chinese hamster cells 4 gene
(XRCC4); A/T c74delA in the gene encoding cytochrome P450
polypeptide CYP3A43; A/C (rs2279115) in the gene encoding B-cell
CLL/lymphoma 2; A/G at +3100 in the 3' UTR (rs2317676) of the gene
encoding Integrin beta 3; -3714 G/T (rs6413429) in the gene
encoding Dopamine transporter 1; A/G (rs 1139417) in the gene
encoding Tumor necrosis factor receptor 1; C/Del (rs1799732) in the
gene encoding Dopamine receptor D2; C/T (rs763110) in the gene
encoding Fas ligand; C/T (rs5743836) in the gene encoding Toll-like
receptor 9; or one or more polymorphisms in linkage disequilibrium
with one or more of these polymorphisms in the assessment of a
subject's risk of developing lung cancer.
30. The use according to claim 29, wherein said use is in
conjunction with the use of at least one further polymorphism
selected from the group consisting of: R19W A/G in the gene
encoding Cerberus 1 (Cer 1); K3326X A/T in the breast cancer 2
early onset gene (BRCA2); V433M A/G in the gene encoding Integrin
alpha-1; E375G T/C in the gene encoding
Calcium/calmodulin-dependent protein kinase kinase 1 (CAMKK1); -81
C/T (rs 2273953) in the 5' UTR of the gene encoding Tumor protein
P73 (P73); or one or more polymorphisms in linkage disequilibrium
with any one of said polymorphisms.
31. The use according to claim 29 or 30 wherein said use is of each
of the polymorpyisms selected from the group consisting of: -133
G/C (rs360721) in the promoter of the gene encoding Interleukin-18;
-251 A/T (rs4073) in the gene encoding Interleukin-8; Arg 197 Gln
(rs 1799930) in the gene encoding N-acetylcysteine transferase 2;
Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the gene
encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; or one or more
polymorphisms in linkage disequilibrium with any one or more of
these polymorphisms.
32. The use according to claim 29 or 30 wherein said use is of each
of the polymorpyisms selected from the group consisting of: -133
G/C (rs360721) in the promoter of the gene encoding Interleukin-18;
-251 A/T (rs4073) in the gene encoding Interleukin-8; Arg 197 Gln
(rs 1799930) in the gene encoding N-acetylcysteine transferase 2;
Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the gene
encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115) in the
gene encoding BCL2; or one or more polymorphisms in linkage
disequilibrium with any one or more of these polymorphisms.
33. The use according to claim 29 or 30 wherein said use is of each
of the polymorpyisms selected from the group consisting of: -133
G/C (rs360721) in the promoter of the gene encoding Interleukin-18;
-251 A/T (rs4073) in the gene encoding Interleukin-8; Arg 197 Gln
(rs 1799930) in the gene encoding N-acetylcysteine transferase 2;
Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; -3714 G/T (rs6413429) in the gene
encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; Arg 312 Gln (rs1799895) in the gene encoding SOD3;
A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding ITGB3;
C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115) in the
gene encoding BCL2; V433M A/G (rs2306022) in the gene encoding
ITGA11; or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
34. The use according to claim 29 or 30 wherein said use is of each
of the polymorpyisms selected from the group consisting of: Rsa 1
C/T (rs2031920) in the gene encoding CYP 2E1; -133 G/C (rs360721)
in the promoter of the gene encoding Interleukin-18; -251 A/T
(rs4073) in the gene encoding Interleukin-8; -511 A/G (rs 16944) in
the gene encoding Interleukin 1B; V433M A/G (rs2306022) in the gene
encoding ITGA11; Arg 197 Gln A/G (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in the gene
encoding .alpha.1-antichymotrypsin; R19W A/G (rs 10115703) in the
gene encoding Cerberus 1; -3714 G/T (rs6413429) in the gene
encoding DAT 1; A/G (rs1139417) in the gene encoding TNFR1; C/T
(rs5743836) in the gene encoding TLR9; -81 C/T (rs 2273953) in the
5' UTR of the gene encoding P73; Arg 312 Gln (rs 1799895) in the
gene encoding SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the
gene encoding ITGB3; C/Del (rs1799732) in the gene encoding DRD2;
A/C (rs2279115) in the gene encoding BCL2; -751 G/T (rs 13181) in
the promoter of the gene encoding XPD; Phe 257 Ser C/T (rs3087386)
in the gene encoding REV1; C/T (rs763110) in the gene encoding
FasL; or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
35. A method of treating a subject having an increased risk of
developing lung cancer comprising the step of replicating,
genotypically or phenotypically, the presence and/or functional
effect of a protective polymorphism selected from the group defined
in claim 11 in said subject.
36. A method of treating a subject having an increased risk of
developing lung cancer, said subject having a detectable
susceptibility polymorphism selected from the group defined in
claim 12 which either upregulates or down-regulates expression of a
gene such that the physiologically active concentration of the
expressed gene product is outside a range which is normal for the
age and sex of the subject, said method comprising the step of
restoring the physiologically active concentration of said product
of gene expression to be within a range which is normal for the age
and sex of the subject.
37. A method of determining a subject's risk of developing lung
cancer, comprising the analysis of two or more polymorphisms
selected from the group consisting of: Ser307Ser G/T in the X-ray
repair complementing defective repair in Chinese hamster cells 4
gene (XRCC4) R19W A/G in the gene encoding Cerberus 1 (Cer 1);
K3326X A/T in the breast cancer 2 early onset gene (BRCA2); V433M
A/G in the gene encoding Integrin alpha-11; or E375G T/C in the
gene encoding Calcium/calmodulin-dependent protein kinase kinase 1
(CAMKK1); A/T c74delA in the gene encoding cytochrome P450
polypeptide CYP3A43; A/C (rs2279115) in the gene encoding B-cell
CLL/lymphoma 2; A/G at +3100 in the 3' UTR (rs2317676) of the gene
encoding Integrin beta 3; -3714 G/T (rs6413429) in the gene
encoding Dopamine transporter 1; A/G (rs1139417) in the gene
encoding Tumor necrosis factor receptor 1; C/Del (rs1799732) in the
gene encoding Dopamine receptor D2; C/T (rs763110) in the gene
encoding Fas ligand; C/T (rs5743836) in the gene encoding Toll-like
receptor 9; -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
Tumor protein P73; or one or more polymorphisms in linkage
disequilibrium with any one or more of these polymorphisms.
38. An antibody microarray for use in the methods as claimed in any
one of claims 1 to 21 or claim 37, which microarray comprises a
substrate presenting antibodies capable of binding to a product of
expression of a gene the expression of which is upregulated or
downregulated when associated with a susceptibility or protective
polymorphism as defined in any one of claims 1 to 5.
39. A method for screening for compounds that modulate the
expression and/or activity of a gene, the expression of which is
upregulated or down-regulated when associated with a susceptibility
or protective polymorphism selected from the group defined in any
one of claims 1 to 5, said method comprising the steps of:
contacting a candidate compound with a cell comprising a
susceptibility or protective polymorphism which has been determined
to be associated with the upregulation or downregulation of
expression of a gene; and measuring the expression of said gene
following contact with said candidate compound, wherein a change in
the level of expression after the contacting step as compared to
before the contacting step is indicative of the ability of the
compound to modulate the expression and/or activity of said
gene.
40. A method according to claim 39 wherein said cell is a human
lung cell which has been pre-screened to confirm the presence of
said polymorphism.
41. A method according to claim 39 or 40 wherein said cell
comprises a susceptibility polymorphism associated with
upregulation of expression of said gene and said screening is for
candidate compounds which down-regulate expression of said
gene.
42. A method according to claim 39 or 40 wherein said cell
comprises a susceptibility polymorphism associated with
downregulation of expression of said gene and said screening is for
candidate compounds which upregulate expression of said gene.
43. A method according to claim 39 or 40 wherein said cell
comprises a protective polymorphism associated with upregulation of
expression of said gene and said screening is for candidate
compounds which further upregulate expression of said gene.
44. A method according to claim 39 or 40 wherein said cell
comprises a protective polymorphism associated with downregulation
of expression of said gene and said screening is for candidate
compounds which further down-regulate expression of said gene.
45. A method for screening for compounds that modulate the
expression and/or activity of a gene, the expression of which is
upregulated or down-regulated when associated with a susceptibility
or protective polymorphism selected from the group defined in any
one of claims 1 to 5, said method comprising the steps of:
contacting a candidate compound with a cell comprising a gene, the
expression of which is upregulated or downregulated when associated
with a susceptibility or protective polymorphism but which in said
cell the expression of which is neither upregulated nor
downregulated; and measuring the expression of said gene following
contact with said candidate compound, wherein a change in the level
of expression after the contacting step as compared to before the
contacting step is indicative of the ability of the compound to
modulate the expression and/or activity of said gene.
46. A method according to claim 45 wherein said cell is a human
lung cell which has been pre-screened to confirm the presence, and
baseline level of expression, of said gene.
47. A method according to claim 45 or 46 wherein expression of the
gene is downregulated when associated with a susceptibility
polymorphism and said screening is for candidate compounds which,
in said cell, upregulate expression of said gene.
48. A method according to claim 45 or 46 wherein expression of the
gene is upregulated when associated with a susceptibility
polymorphism and said screening is for candidate compounds which,
in said cell, down-regulate expression of said gene.
49. A method according to claim 45 or 46 wherein expression of the
gene is upregulated when associated with a protective polymorphism
and said screening is for compounds which, in said cell, upregulate
expression of said gene.
50. A method according to claim 45 or 46 wherein expression of the
gene is downregulated when associated with a protective
polymorphism and said screening is for compounds which, in said
cell, downregulate expression of said gene.
51. A method of assessing the likely responsiveness of a subject
predisposed to or diagnosed with lung cancer to a prophylactic or
therapeutic treatment, which treatment involves restoring the
physiologically active concentration of a product of gene
expression to be within a range which is normal for the age and sex
of the subject, which method comprises detecting in said subject
the presence or absence of a susceptibility polymorphism selected
from the group defined in claim 1 which when present either
upregulates or down-regulates expression of said gene such that the
physiological active concentration of the expressed gene product is
outside said normal range, wherein the detection of the presence of
said polymorphism is indicative of the subject likely responding to
said treatment.
52. A method of assessing a subject's suitability for an
intervention diagnostic of or therapeutic for lung cancer, the
method comprising: a) providing a net score for said subject,
wherein the net score is or has been determined by: i) providing
the result of one or more genetic tests of a sample from the
subject, and analysing the result for the presence or absence of
protective polymorphisms and for the presence or absence of
susceptibility polymorphisms, wherein said protective and
susceptibility polymorphisms are associated with lung cancer, ii)
assigning a positive score for each protective polymorphism and a
negative score for each susceptibility polymorphism or vice versa;
iii) calculating a net score for said subject by representing the
balance between the combined value of the protective polymorphisms
and the combined value of the susceptibility polymorphisms present
in the subject sample; and b) providing a distribution of net
scores for lung cancer sufferers and non-sufferers wherein the net
scores for lung cancer sufferers and non-sufferers are or have been
determined in the same manner as the net score determined for said
subject; and c) determining whether the net score for said subject
lies within a threshold on said distribution separating individuals
deemed suitable for said intervention from those for whom said
intervention is deemed unsuitable; wherein a net score within said
threshold is indicative of the subject's suitability for the
intervention, and wherein a net score outside the threshold is
indicative of the subject's unsuitability for the intervention.
53. The method according to claim 52 wherein the value assigned to
each protective polymorphism is the same.
54. The method according to any one of claims 52 to 53 wherein the
value assigned to each susceptibility polymorphism is the same.
55. The method according any one of claims 52 to 54 wherein the
intervention is a diagnostic test for lung cancer.
56. The method according to any one of claims 52 to 54 wherein
intervention is a therapeutic intervention for lung cancer.
57. The method according to claim 52 wherein the lung cancer is
selected from the group consisting of non-small cell lung cancer
including adenocarcinoma and squamous cell carcinoma, small cell
lung cancer, carcinoid tumor, lymphoma, or metastatic cancer.
58. The method according to claim 52 wherein the protective and
susceptibility polymorphisms are selected from the group consisting
of: the -133 G/C polymorphism in the Interleukin-18 gene; the -1053
C/T polymorphism in the CYP 2E1 gene; the Arg197gln polymorphism in
the Nat2 gene; the -511 G/A polymorphism in the Interleukin IB
gene; the Ala 9 Thr polymorphism in the Anti-chymotrypsin gene; the
S allele polymorphism in the Alpha1-antitrypsin gene; the -251 A/T
polymorphism in the Interleukin-8 gene; the Lys 751 gln
polymorphism in the XPD gene; the +760 G/C polymorphism in the SOD3
gene; the Phe257Ser polymorphism in the REV gene; the Z alelle
polymorphism in the Alpha1-antitrypsin gene; the R19W A/G
polymorphism in the Cerberus 1 (Cer 1) gene; the Ser307Ser G/T
polymorphism in the XRCC4 gene; the K3326X A/T polymorphism in the
BRCA2 gene; the V433M A/G polymorphism in the Integrin alpha-11
gene; the E375G T/C polymorphism in the CAMKK1 gene; the A/T
c74delA polymorphism in the gene encoding cytochrome P450
polypeptide CYP3A43; the A/C (rs2279115) polymorphism in the gene
encoding B-cell CLL/lymphoma 2; the A/G at +3100 in the 3' UTR
(rs2317676) polymorphism of the gene encoding Integrin beta 3; the
-3714 G/T (rs6413429) polymorphism in the gene encoding Dopamine
transporter 1; the A/G (rs1139417) polymorphism in the gene
encoding Tumor necrosis factor receptor 1; the C/Del (rs1799732)
polymorphism in the gene encoding Dopamine receptor D2; the C/T
(rs763110) polymorphism in the gene encoding Fas ligand; the C/T
(rs5743836) polymorphism in the gene encoding Toll-like receptor 9;
the -81 C/T (rs 2273953) polymorphism in the 5' UTR of the gene
encoding Tumor protein P73; or one or more polymorphisms in linkage
disequilibrium with one or more of said polymorphisms.
59. The method according to claim 40 wherein the result is analysed
for the presence of absence of each of the polymorphisms from the
group consisting of: -133 G/C (rs360721) in the promoter of the
gene encoding Interleukin-18; -251 A/T (rs4073) in the gene
encoding Interleukin-8; Arg 197 Gln (rs 1799930) in the gene
encoding N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in
the gene encoding .alpha.1-antichymotrypsin; -3714 G/T (rs6413429)
in the gene encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of
the gene encoding P73; Arg 312 Gln (rs1799895) in the gene encoding
SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding
ITGB3; C/Del (rs1799732) in the gene encoding DRD2; or one or more
polymorphisms in linkage disequilibrium with any one or more of
these polymorphisms.
60. The method according to claim 40 wherein the result is analysed
for the presence of absence of each of the polymorphisms from the
group consisting of: -133 G/C (rs360721) in the promoter of the
gene encoding Interleukin-18; -251 A/T (rs4073) in the gene
encoding Interleukin-8; Arg 197 Gln (rs 1799930) in the gene
encoding N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in
the gene encoding .alpha.1-antichymotrypsin; -3714 G/T (rs6413429)
in the gene encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of
the gene encoding P73; Arg 312 Gln (rs1799895) in the gene encoding
SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding
ITGB3; C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115)
in the gene encoding BCL2; or one or more polymorphisms in linkage
disequilibrium with any one or more of these polymorphisms.
61. The method according to claim 40 wherein the result is analysed
for the presence of absence of each of the polymorphisms from the
group consisting of: -133 G/C (rs360721) in the promoter of the
gene encoding Interleukin-18; -251 A/T (rs4073) in the gene
encoding Interleukin-8; Arg 197 Gln (rs 1799930) in the gene
encoding N-acetylcysteine transferase 2; Ala 15 Thr A/G (rs4934) in
the gene encoding .alpha.1-antichymotrypsin; -3714 G/T (rs6413429)
in the gene encoding DAT 1; -81 C/T (rs 2273953) in the 5' UTR of
the gene encoding P73; Arg 312 Gln (rs1799895) in the gene encoding
SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding
ITGB3; C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115)
in the gene encoding BCL2; V433M A/G (rs2306022) in the gene
encoding ITGA11; or one or more polymorphisms in linkage
disequilibrium with any one or more of these polymorphisms.
62. The method according to claim 40 wherein the result is analysed
for the presence of absence of each of the polymorphisms from the
group consisting of: Rsa 1 C/T (rs2031920) in the gene encoding CYP
2E1; -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18; -251 A/T (rs4073) in the gene encoding
Interleukin-8; -511 A/G (rs 16944) in the gene encoding Interleukin
1B; V433M A/G (rs2306022) in the gene encoding ITGA11; Arg 197 Gln
A/G (rs 1799930) in the gene encoding N-acetylcysteine transferase
2; Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; R19W A/G (rs 10115703) in the gene
encoding Cerberus 1; -3714 G/T (rs6413429) in the gene encoding DAT
1; A/G (rs1139417) in the gene encoding TNFR1; C/T (rs5743836) in
the gene encoding TLR9; -81 C/T (rs 2273953) in the 5' UTR of the
gene encoding P73; Arg 312 Gln (rs 1799895) in the gene encoding
SOD3; A/G at +3100 in the 3' UTR (rs2317676) of the gene encoding
ITGB3; C/Del (rs1799732) in the gene encoding DRD2; A/C (rs2279115)
in the gene encoding BCL2; -751 G/T (rs 13181) in the promoter of
the gene encoding XPD; Phe 257 Ser C/T (rs3087386) in the gene
encoding REV1; C/T (rs763110) in the gene encoding FasL; or one or
more polymorphisms in linkage disequilibrium with any one or more
of these polymorphisms.
63. The method according to claim 57 or 58 wherein said
intervention is a CT scan for lung cancer.
64. The method according to any one of claims 52 to 58 as described
herein with reference to the examples and/or figures.
65. A kit for assessing a subject's risk of developing one or more
obstructive lung diseases selected from lung cancer, said kit
comprising a means of analysing a sample from said subject for the
presence or absence of one or more polymorphisms selected from the
group consisting of: Ser307Ser G/T polymorphism in the X-ray repair
complementing defective repair in Chinese hamster cells 4 gene
(XRCC4); A/T c74delA in the gene encoding cytochrome P450
polypeptide CYP3A43; A/C (rs2279115) in the gene encoding B-cell
CLL/lymphoma 2; A/G at +3100 in the 3' UTR (rs2317676) of the gene
encoding Integrin beta 3; -3714 G/T (rs6413429) in the gene
encoding Dopamine transporter 1; A/G (rs 1139417) in the gene
encoding Tumor necrosis factor receptor 1; C/Del (rs1799732) in the
gene encoding Dopamine receptor D2; C/T (rs763110) in the gene
encoding Fas ligand; C/T (rs5743836) in the gene encoding Toll-like
receptor 9; or one or more polymorphisms which are in linkage
disequilibrium with one or more of these polymorphisms.
Description
FIELD OF THE INVENTION
[0001] The present invention is concerned with methods for
assessment of pulmonary function and/or disorders, and in
particular for assessing risk of developing lung cancer in smokers
and non-smokers using analysis of genetic polymorphisms.
BACKGROUND OF THE INVENTION
[0002] Lung cancer is the second most common cancer and has been
attributed primarily to cigarette smoking. Other factors
contributing to the development of lung cancer include occupational
exposure, genetic factors, radon exposure, exposure to other
aero-pollutants and possibly dietary factors (Alberg A J, et al.,
2003). Non-smokers are estimated to have a one in 400 risk of lung
cancer (0.25%). Smoking increases this risk by approximately 40
fold, such that smokers have a one in 10 risk of lung cancer (10%)
and in long-term smokers the life-time risk of lung cancer has been
reported to be as high 10-15% (Schwartz A G. 2004). Genetic factors
are thought to play some part as evidenced by a weak familial
tendency (among smokers) and the fact that only the minority of
smokers get lung cancer. It is generally accepted that the majority
of this genetic tendency comes from low penetrant high frequency
polymorphisms, that is, polymorphisms which are common in the
general population that in context of chronic smoking exposure
contribute collectively to cancer development (Schwartz A G. 2004,
Wu X et al., 2004). Several epidemiological studies have reported
that impaired lung function (Anthonisen N R. 1989, Skillrud D M.
1986, Tockman M S et al., 1987, Kuller L H, et al., 1990, Nomura A,
et al., 1991) or symptoms of obstructive lung disease (Mayne S T,
et al., 1999) are independent risk factors for lung cancer and are
possibly more relevant than smoking exposure dose.
[0003] Despite advances in the treatment of airways disease,
current therapies do not significantly alter the natural history of
lung cancer, which may include metastasis and progressive loss of
lung function causing respiratory failure and death. Although
cessation of smoking may be expected to reduce this decline in lung
function, it is probable that if this is not achieved at an early
stage, the loss is considerable and symptoms of worsening
breathlessness likely cannot be averted. Analogous to the discovery
of serum cholesterol and its link to coronary artery disease, there
is a need to better understand the factors that contribute to lung
cancer so that tests that identify at risk subjects can be
developed and that new treatments can be discovered to reduce the
adverse effects of lung cancer. The early diagnosis of lung cancer
or of a propensity to developing lung cancer enables a broader
range of prophylactic or therapeutic treatments to be employed than
can be employed in the treatment of late stage lung cancer. Such
prophylactic or early therapeutic treatment is also more likely to
be successful, achieve remission, improve quality of life, and/or
increase lifespan.
[0004] To date, a number of biomarkers useful in the diagnosis and
assessment of propensity towards developing various pulmonary
disorders have been identified. These include, for example, single
nucleotide polymorphisms including the following: A-82G in the
promoter of the gene encoding human macrophage elastase (MMP12);
T.fwdarw.C within codon 10 of the gene encoding transforming growth
factor beta (TGF.beta.); C+760G of the gene encoding superoxide
dismutase 3 (SOD3); T-1296C within the promoter of the gene
encoding tissue inhibitor of metalloproteinase 3 (TIMP3); and
polymorphisms in linkage disequilibrium with these polymorphisms,
as disclosed in PCT International Application PCT/NZ02/00106
(published as WO 02/099134 and incorporated herein in its
entirety).
[0005] It would be desirable and advantageous to have additional
biomarkers which could be used to assess a subject's risk of
developing pulmonary disorders such as lung cancer, or a risk of
developing lung cancer-related impaired lung function, particularly
if the subject is a smoker.
[0006] It is primarily to such biomarkers and their use in methods
to assess risk of developing such disorders that the present
invention is directed.
SUMMARY OF THE INVENTION
[0007] The present invention is primarily based on the finding that
certain polymorphisms are found more often in subjects with lung
cancer than in control subjects. Analysis of these polymorphisms
reveals an association between polymorphisms and the subject's risk
of developing lung cancer.
[0008] Thus, according to one aspect there is provided a method of
determining a subject's risk of developing lung cancer comprising
analysing a sample from said subject for the presence or absence of
one or more polymorphisms selected from the group consisting of:
[0009] Ser307Ser G/T (rs1056503) in the X-ray repair complementing
defective repair in Chinese hamster cells 4 gene (XRCC4), [0010]
A/T c74delA in the gene encoding cytochrome P450 polypeptide
CYP3A43 (CYP3A43), [0011] A/C (rs2279115) in the gene encoding
B-cell CLL/lymphoma 2 (BCL2), [0012] A/G at +3100 in the 3'UTR
(rs2317676) of the gene encoding Integrin beta 3 (ITGB3), [0013]
-3714 G/T (rs6413429) in the gene encoding Dopamine transporter 1
(DAT1), [0014] A/G (rs1139417) in the gene encoding Tumor necrosis
factor receptor 1 (TNFR1), [0015] C/Del (rs1799732) in the gene
encoding Dopamine receptor D2 (DRD2), [0016] C/T (rs763110) in the
gene encoding Fas ligand (FasL), or [0017] C/T (rs5743836) in the
gene encoding Toll-like receptor 9 (TLR9),
[0018] wherein the presence or absence of said polymorphism is
indicative of the subject's risk of developing lung cancer.
[0019] This polymorphism can be detected directly or by detection
of one or more polymorphisms which are in linkage disequilibrium
with one or more of said polymorphisms.
[0020] Linkage disequilibrium (LD) is a phenomenon in genetics
whereby two or more mutations or polymorphisms are in such close
genetic proximity that they are co-inherited. This means that in
genotyping, detection of one polymorphism as present infers the
presence of the other. (Reich D E et al; Linkage disequilibrium in
the human genome, Nature 2001, 411:199-204.)
[0021] The lung cancer may be non-small cell lung cancer including
adenocarcinoma and squamous cell carcinoma, or small cell lung
cancer, or may be a carcinoid tumor, a lymphoma, or a metastatic
cancer.
[0022] The method can additionally comprise analysing a sample from
said subject for the presence or absence of one or more further
polymorphisms selected from the group consisting of: [0023] R19W
A/G (rs10115703) in the gene encoding Cerberus 1 (Cer 1); [0024]
K3326X A/T (rs 1571833) in the breast cancer 2 early onset gene
(BRCA2); [0025] V433M A/G (rs2306022) in the gene encoding Integrin
alpha-1; [0026] E375G T/C (rs7214723) in the gene encoding
Calcium/calmodulin-dependent protein kinase kinase 1 (CAMKK1); or
[0027] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
Tumor protein P73 (P73).
[0028] Again, detection of the one or more further polymorphisms
may be carried out directly or by detection of polymorphisms in
linkage disequilibrium with the one or more further
polymorphisms.
[0029] The presence of one or more polymorphisms selected from the
group consisting of:
[0030] the E375G T/C TT genotype in the gene encoding CAMKK1;
[0031] the -81 C/T (rs 2273953) CC genotype the gene encoding
P73;
[0032] the A/C (rs2279115) AA genotype in the gene encoding
BCL2;
[0033] the +3100 A/G (rs2317676) AG or GG genotype in the gene
encoding ITGB3;
[0034] the C/Del (rs1799732) CDel or DelDel genotype in the gene
encoding DRD2; or
[0035] the C/T (rs763110) TT genotype in the gene encoding
FasL,
may be indicative of a reduced risk of developing lung cancer.
[0036] The presence of one or more polymorphisms selected from the
group consisting of:
[0037] the R19W A/G AA or GG genotype in the gene encoding Cer
1;
[0038] the Ser307Ser G/T GG or GT genotype in the XRCC4 gene;
[0039] the K3326X A/T AT or TT genotype in the BRCA2 gene;
[0040] the V433M A/G AA genotype in the gene encoding Integrin
alpha-11;
[0041] the A/T c74delA AT or TT genotype in the gene encoding
CYP3A43;
[0042] the -3714 G/T (rs6413429) GT or TT genotype in the gene
encoding DAT 1;
[0043] the A/G (rs1139417) AA genotype in the gene encoding TNFR1;
or
[0044] the C/T (rs5743836) CC genotype in the gene encoding
TLR9,
may be indicative of an increased risk of developing lung
cancer.
[0045] The methods of the invention are particularly useful in
smokers (both current and former).
[0046] It will be appreciated that the methods of the invention
identify two categories of polymorphisms--namely those associated
with a reduced risk of developing lung cancer (which can be termed
"protective polymorphisms") and those associated with an increased
risk of developing lung cancer (which can be termed "susceptibility
polymorphisms").
[0047] Therefore, the present invention further provides a method
of assessing a subject's risk of developing lung cancer, said
method comprising:
[0048] determining the presence or absence of at least one
protective polymorphism associated with a reduced risk of
developing lung cancer; and
[0049] in the absence of at least one protective polymorphism,
determining the presence or absence of at least one susceptibility
polymorphism associated with an increased risk of developing lung
cancer;
[0050] wherein the presence of one or more of said protective
polymorphisms is indicative of a reduced risk of developing lung
cancer, and the absence of at least one protective polymorphism in
combination with the presence of at least one susceptibility
polymorphism is indicative of an increased risk of developing lung
cancer.
[0051] Preferably, the at least one protective polymorphism
selected from the group consisting of:
[0052] the E375G T/C TT genotype in the gene encoding CAMKK1;
[0053] the -81 C/T (rs 2273953) CC genotype the gene encoding
P73;
[0054] the A/C (rs2279115) AA genotype in the gene encoding
BCL2;
[0055] the +3100 A/G (rs2317676) AG or GG genotype in the gene
encoding ITGB3;
[0056] the C/Del (rs1799732) CDel or DelDel genotype in the gene
encoding DRD2; or
[0057] the C/T (rs763110) TT genotype in the gene encoding Fas
ligand.
[0058] The at least one susceptibility polymorphism may be selected
from the group consisting of:
[0059] the R19W A/G AA or GG genotype in the gene encoding Cer
1;
[0060] the Ser307Ser G/T GG or GT genotype in the XRCC4 gene;
[0061] the K3326X A/T AT or TT genotype in the BRCA2 gene;
[0062] the V433M A/G AA genotype in the gene encoding Integrin
alpha-1;
[0063] the A/T c74delA AT or TT genotype in the gene encoding
CYP3A43;
[0064] the -3714 G/T (rs6413429) GT or TT genotype in the gene
encoding DAT 1;
[0065] the A/G (rs1139417) AA genotype in the gene encoding TNFR1;
or
[0066] the C/T (rs5743836) CC genotype in the gene encoding
TLR9.
[0067] In a preferred form of the invention the presence of two or
more protective polymorphisms is indicative of a reduced risk of
developing lung cancer.
[0068] In a further preferred form of the invention the presence of
two or more susceptibility polymorphisms is indicative of an
increased risk of developing lung cancer.
[0069] In still a further preferred form of the invention the
presence of two or more protective polymorphisms irrespective of
the presence of one or more susceptibility polymorphisms is
indicative of reduced risk of developing lung cancer.
[0070] In another aspect, the invention provides a method of
determining a subject's risk of developing lung cancer, said method
comprising obtaining the result of one or more genetic tests of a
sample from said subject, and analysing the result for the presence
or absence of one or more polymorphisms selected from the group
consisting of: [0071] Ser307Ser G/T in the X-ray repair
complementing defective repair in Chinese hamster cells 4 gene;
[0072] A/T c74delA in the gene encoding cytochrome P450 polypeptide
CYP3A43, [0073] A/C (rs2279115) in the gene encoding B-cell
CLL/lymphoma 2, [0074] A/G at +3100 in the 3'UTR (rs2317676) of the
gene encoding Integrin beta 3, [0075] -3714 G/T (rs6413429) in the
gene encoding Dopamine transporter 1, [0076] A/G (rs1139417) in the
gene encoding Tumor necrosis factor receptor 1,
[0077] C/Del (rs1799732) in the gene encoding Dopamine receptor
D2,
[0078] C/T (rs763110) in the gene encoding Fas ligand,
[0079] C/T (rs5743836) in the gene encoding Toll-like receptor
9,
[0080] or one or more polymorphisms in linkage disequilibrium with
this polymorphism;
[0081] wherein a result indicating the presence or absence of one
or more of said polymorphisms is indicative of the subject's risk
of developing lung cancer.
[0082] The method can additionally comprise obtaining the result of
one or more genetic tests of a sample from said subject, and
analysing the result for the presence or absence of one or more
further polymorphisms selected from the group consisting of: [0083]
R19W A/G in the gene encoding Cerberus 1; [0084] K3326X A/T in the
breast cancer 2 early onset gene; [0085] V433M A/G in the gene
encoding Integrin alpha-1; [0086] E375G T/C in the gene encoding
Calcium/calmodulin-dependent protein kinase kinase 1; or [0087] -81
C/T (rs 2273953) in the 5' UTR of the gene encoding Tumor protein
P73.
[0088] Again, the presence or absence may be determined directly or
by determining the presence or absence of polymorphisms in linkage
disequilibrium with the one or more further polymorphisms.
[0089] In a further aspect there is provided a method of
determining a subject's risk of developing lung cancer comprising
the analysis of two or more polymorphisms selected from the group
consisting of: [0090] R19W A/G in the gene encoding Cerberus 1;
[0091] Ser307Ser G/T in the X-ray repair complementing defective
repair in Chinese hamster cells 4 gene; [0092] K3326X A/T in the
breast cancer 2 early onset gene; [0093] V433M A/G in the gene
encoding Integrin alpha-11; or [0094] E375G T/C in the gene
encoding Calcium/calmodulin-dependent protein kinase kinase 1;
[0095] A/T c74delA in the gene encoding cytochrome P450 polypeptide
CYP3A43, [0096] A/C (rs2279115) in the gene encoding B-cell
CLL/lymphoma 2, [0097] A/G at +3100 in the 3'UTR (rs2317676) of the
gene encoding Integrin beta 3, [0098] -3714 G/T (rs6413429) in the
gene encoding Dopamine transporter 1, [0099] A/G (rs1139417) in the
gene encoding Tumor necrosis factor receptor 1, [0100] C/Del
(rs1799732) in the gene encoding Dopamine receptor D2, [0101] C/T
(rs763110) in the gene encoding Fas ligand, [0102] C/T (rs5743836)
in the gene encoding Toll-like receptor 9, [0103] -81 C/T (rs
2273953) in the 5' UTR of the gene encoding Tumor protein P73,
or
[0104] one or more polymorphisms in linkage disequilibrium with any
one or more of these polymorphisms.
[0105] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0106] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0107] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0108] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0109] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0110] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0111] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0112] Arg 312 Gln (rs 1799895) in the gene encoding SOD3;
[0113] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0114] C/Del (rs1799732) in the gene encoding DRD2;
[0115] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0116] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0117] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0118] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0119] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0120] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0121] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0122] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0123] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0124] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0125] C/Del (rs1799732) in the gene encoding DRD2;
[0126] A/C (rs2279115) in the gene encoding BCL2;
[0127] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0128] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0129] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0130] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0131] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0132] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0133] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0134] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0135] Arg 312 Gln (rs 1799895) in the gene encoding SOD3;
[0136] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0137] C/Del (rs1799732) in the gene encoding DRD2;
[0138] A/C (rs2279115) in the gene encoding BCL2;
[0139] V433M A/G (rs2306022) in the gene encoding ITGA11;
[0140] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0141] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected: [0142]
Rsa 1 C/T (rs2031920) in the gene encoding CYP 2E1; [0143] -133 G/C
(rs360721) in the promoter of the gene encoding Interleukin-18;
[0144] -251 A/T (rs4073) in the gene encoding Interleukin-8; [0145]
-511 A/G (rs 16944) in the gene encoding Interleukin 1B; [0146]
V433M A/G (rs2306022) in the gene encoding ITGA11; [0147] Arg 197
Gln A/G (rs 1799930) in the gene encoding N-acetylcysteine
transferase 2; [0148] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; [0149] R19W A/G (rs 10115703) in the
gene encoding Cerberus 1; [0150] -3714 G/T (rs6413429) in the gene
encoding DAT 1; [0151] A/G (rs1139417) in the gene encoding TNFR1;
[0152] C/T (rs5743836) in the gene encoding TLR9; [0153] -81 C/T
(rs 2273953) in the 5' UTR of the gene encoding P73; [0154] Arg 312
Gln (rs 1799895) in the gene encoding SOD3; [0155] A/G at +3100 in
the 3'UTR (rs2317676) of the gene encoding ITGB3; [0156] C/Del
(rs1799732) in the gene encoding DRD2;
[0157] A/C (rs2279115) in the gene encoding BCL2;
[0158] -751 G/T (rs 13181) in the promoter of the gene encoding
XPD;
[0159] Phe 257 Ser C/T (rs3087386) in the gene encoding REVI;
[0160] C/T (rs763110) in the gene encoding FasL;
[0161] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0162] In various embodiments, any one or more of the above methods
comprises the step of analysing the amino acid present at a
position mapping to codon 19 of the gene encoding Cer 1.
[0163] The presence of tryptophan at said position is indicative of
an increased risk of developing lung cancer.
[0164] The presence of arginine at said position is indicative of
reduced risk of developing lung cancer.
[0165] In various embodiments, any one or more of the above methods
comprises the step of analysing the amino acid present at a
position mapping to codon 3326 in the BRCA2 gene.
[0166] The presence of lysine at said position is indicative of
reduced risk of developing lung cancer.
[0167] The presence of a truncated gene product of 3325 amino acids
is indicative of an increased risk of developing lung cancer.
[0168] In various embodiments, any one or more of the above methods
comprises the step of analysing the amino acid present at a
position mapping to codon 433 in the gene encoding Integrin
alpha-11.
[0169] The presence of methionine at said position is indicative of
an increased risk of developing lung cancer.
[0170] The presence of valine at said position is indicative of
reduced risk of developing lung cancer.
[0171] In various embodiments, any one or more of the above methods
comprises the step of analysing the amino acid present at a
position mapping to codon 375 in the gene encoding CAMKK1.
[0172] The presence of glycine at said position is indicative of an
increased risk of developing lung cancer.
[0173] The presence of glutamate at said position is indicative of
reduced risk of developing lung cancer.
[0174] In a preferred form of the invention the methods as
described herein are performed in conjunction with an analysis of
one or more risk factors, including one or more epidemiological
risk factors, associated with a risk of developing lung cancer.
Such epidemiological risk factors include but are not limited to
smoking or exposure to tobacco smoke, age, sex, and familial
history of lung cancer.
[0175] In a further aspect, the invention provides for the use of
at least one polymorphism in the assessment of a subject's risk of
developing lung cancer, wherein the at least one polymorphism is
selected from the group consisting of, [0176] Ser307Ser G/T in the
X-ray repair complementing defective repair in Chinese hamster
cells 4 gene; [0177] A/T c74delA in the gene encoding cytochrome
P450 polypeptide CYP3A43, [0178] A/C (rs2279115) in the gene
encoding B-cell CLL/lymphoma 2, [0179] A/G at +3100 in the 3'UTR
(rs2317676) of the gene encoding Integrin beta 3, [0180] -3714 G/T
(rs6413429) in the gene encoding Dopamine transporter 1, [0181] A/G
(rs1139417) in the gene encoding Tumor necrosis factor receptor 1,
[0182] C/Del (rs1799732) in the gene encoding Dopamine receptor D2,
[0183] C/T (rs763110) in the gene encoding Fas ligand, or [0184]
C/T (rs5743836) in the gene encoding Toll-like receptor 9,
[0185] or one or more polymorphisms in linkage disequilibrium with
said polymorphism.
[0186] Optionally, said use may be in conjunction with the use of
at least one further polymorphism selected from the group
consisting of: [0187] R19W A/G in the gene encoding Cerberus 1 (Cer
1); [0188] K3326X A/T in the breast cancer 2 early onset gene
(BRCA2); [0189] V433M A/G in the gene encoding Integrin alpha-1;
[0190] E375G T/C in the gene encoding Calcium/calmodulin-dependent
protein kinase kinase 1 (CAMKK1); [0191] -81 C/T (rs 2273953) in
the 5' UTR of the gene encoding Tumor protein P73; [0192] or one or
more polymorphisms which are in linkage disequilibrium with any one
or more of these polymorphisms.
[0193] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0194] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0195] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0196] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0197] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0198] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0199] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0200] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0201] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0202] C/Del (rs1799732) in the gene encoding DRD2;
[0203] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0204] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0205] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0206] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0207] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0208] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0209] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0210] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0211] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0212] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0213] C/Del (rs1799732) in the gene encoding DRD2;
[0214] A/C (rs2279115) in the gene encoding BCL2;
[0215] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0216] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0217] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0218] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0219] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0220] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0221] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0222] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0223] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0224] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0225] C/Del (rs1799732) in the gene encoding DRD2;
[0226] A/C (rs2279115) in the gene encoding BCL2;
[0227] V433M A/G (rs2306022) in the gene encoding ITGA11;
[0228] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0229] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0230] Rsa 1 C/T (rs2031920) in the gene encoding CYP 2E1;
[0231] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0232] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0233] -511 A/G (rs 16944) in the gene encoding Interleukin 1B;
[0234] V433M A/G (rs2306022) in the gene encoding ITGA11;
[0235] Arg 197 Gln A/G (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0236] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0237] R19W A/G (rs 10115703) in the gene encoding Cerberus 1;
[0238] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0239] A/G (rs1139417) in the gene encoding TNFR1;
[0240] C/T (rs5743836) in the gene encoding TLR9;
[0241] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0242] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0243] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0244] C/Del (rs1799732) in the gene encoding DRD2;
[0245] A/C (rs2279115) in the gene encoding BCL2;
[0246] -751 G/T (rs 13181) in the promoter of the gene encoding
XPD;
[0247] Phe 257 Ser C/T (rs3087386) in the gene encoding REV1;
[0248] C/T (rs763110) in the gene encoding FasL;
[0249] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0250] In another aspect the invention provides a set of nucleotide
probes and/or primers for use in the preferred methods of the
invention herein described. Preferably, the nucleotide probes
and/or primers are those which span, or are able to be used to
span, the polymorphic regions of the genes. Also provided are one
or more nucleotide probes and/or primers comprising the sequence of
any one of the probes and/or primers herein described, including
any one comprising the sequence of any one of SEQ.ID.NO. 1 to 72,
more preferably any one of SEQ.ID.NO. 1 to 10 or any one of
SEQ.ID.NO. 26 to 43.
[0251] In yet a further aspect, the invention provides a nucleic
acid microarray for use in the methods of the invention, which
microarray comprises a substrate presenting nucleic acid sequences
capable of hybridizing to nucleic acid sequences which encode one
or more of the susceptibility or protective polymorphisms described
herein or sequences complimentary thereto.
[0252] In another aspect, the invention provides an antibody
microarray for use in the methods of the invention, which
microarray comprises a substrate presenting antibodies capable of
binding to a product of expression of a gene the expression of
which is upregulated or downregulated when associated with a
susceptibility or protective polymorphism as described herein.
[0253] In a further aspect the present invention provides a method
treating a subject having an increased risk of developing lung
cancer comprising the step of replicating, genotypically or
phenotypically, the presence and/or functional effect of a
protective polymorphism in said subject.
[0254] In yet a further aspect, the present invention provides a
method of treating a subject having an increased risk of developing
lung cancer, said subject having a detectable susceptibility
polymorphism which either upregulates or down-regulates expression
of a gene such that the physiologically active concentration of the
expressed gene product is outside a range which is normal for the
age and sex of the subject, said method comprising the step of
restoring the physiologically active concentration of said product
of gene expression to be within a range which is normal for the age
and sex of the subject.
[0255] In yet a further aspect, the present invention provides a
method for screening for compounds that modulate the expression
and/or activity of a gene, the expression of which is upregulated
or downregulated when associated with a susceptibility or
protective polymorphism, said method comprising the steps of:
[0256] contacting a candidate compound with a cell comprising a
susceptibility or protective polymorphism which has been determined
to be associated with the upregulation or downregulation of
expression of a gene; and
[0257] measuring the expression of said gene following contact with
said candidate compound,
[0258] wherein a change in the level of expression after the
contacting step as compared to before the contacting step is
indicative of the ability of the compound to modulate the
expression and/or activity of said gene.
[0259] Preferably, said cell is a human lung cell which has been
pre-screened to confirm the presence of said polymorphism.
[0260] Preferably, said cell comprises a susceptibility
polymorphism associated with upregulation of expression of said
gene and said screening is for candidate compounds which
downregulate expression of said gene.
[0261] Alternatively, said cell comprises a susceptibility
polymorphism associated with downregulation of expression of said
gene and said screening is for candidate compounds which upregulate
expression of said gene.
[0262] In another embodiment, said cell comprises a protective
polymorphism associated with upregulation of expression of said
gene and said screening is for candidate compounds which further
upregulate expression of said gene.
[0263] Alternatively, said cell comprises a protective polymorphism
associated with downregulation of expression of said gene and said
screening is for candidate compounds which further downregulate
expression of said gene.
[0264] In another aspect, the present invention provides a method
for screening for compounds that modulate the expression and/or
activity of a gene, the expression of which is upregulated or
downregulated when associated with a susceptibility or protective
polymorphism, said method comprising the steps of:
[0265] contacting a candidate compound with a cell comprising a
gene, the expression of which is upregulated or downregulated when
associated with a susceptibility or protective polymorphism but
which in said cell the expression of which is neither upregulated
nor downregulated; and
[0266] measuring the expression of said gene following contact with
said candidate compound,
[0267] wherein a change in the level of expression after the
contacting step as compared to before the contacting step is
indicative of the ability of the compound to modulate the
expression and/or activity of said gene.
[0268] Preferably, expression of the gene is downregulated when
associated with a susceptibility polymorphism once said screening
is for candidate compounds which in said cell, upregulate
expression of said gene.
[0269] Preferably, said cell is a human lung cell which has been
pre-screened to confirm the presence, and baseline level of
expression, of said gene.
[0270] Alternatively, expression of the gene is upregulated when
associated with a susceptibility polymorphism and said screening is
for candidate compounds which, in said cell, downregulate
expression of said gene.
[0271] In another embodiment, expression of the gene is upregulated
when associated with a protective polymorphism and said screening
is for compounds which, in said cell, upregulate expression of said
gene.
[0272] Alternatively, expression of the gene is downregulated when
associated with a protective polymorphism and said screening is for
compounds which, in said cell, downregulate expression of said
gene.
[0273] In yet a further aspect, the present invention provides a
method of assessing the likely responsiveness of a subject at risk
of developing or suffering from lung cancer to a prophylactic or
therapeutic treatment, which treatment involves restoring the
physiologically active concentration of a product of gene
expression to be within a range which is normal for the age and sex
of the subject, which method comprises detecting in said subject
the presence or absence of a susceptibility polymorphism which when
present either upregulates or downregulates expression of said gene
such that the physiological active concentration of the expressed
gene product is outside said normal range, wherein the detection of
the presence of said polymorphism is indicative of the subject
likely responding to said treatment.
[0274] In still a further aspect, the present invention provides a
method of assessing a subject's suitability for an intervention
that is diagnostic of or therapeutic for a disease, the method
comprising:
[0275] a) providing a net score for said subject, wherein the net
score is or has been determined by: [0276] i) providing the result
of one or more genetic tests of a sample from the subject, and
analysing the result for the presence or absence of protective
polymorphisms and for the presence or absence of susceptibility
polymorphisms, wherein said protective and susceptibility
polymorphisms are associated with said disease, [0277] ii)
assigning a positive score for each protective polymorphism and a
negative score for each susceptibility polymorphism or vice versa;
[0278] iii) calculating a net score for said subject by
representing the balance between the combined value of the
protective polymorphisms and the combined value of the
susceptibility polymorphisms present in the subject sample; and
[0279] b) providing a distribution of net scores for disease
sufferers and non-sufferers wherein the net scores for disease
sufferers and non-sufferers are or have been determined in the same
manner as the net score determined for said subject;
[0280] c) determining whether the net score for said subject lies
within a threshold on said distribution separating individuals
deemed suitable for said intervention from those for whom said
intervention is deemed unsuitable;
[0281] wherein a net score within said threshold is indicative of
the subject's suitability for the intervention, and wherein a net
score outside the threshold is indicative of the subject's
unsuitability for the intervention.
[0282] The value assigned to each protective polymorphism may be
the same or may be different. The value assigned to each
susceptibility polymorphism may be the same or may be different,
with either each protective polymorphism having a negative value
and each susceptibility polymorphism having a positive value, or
vice versa.
[0283] In one embodiment, the intervention is a diagnostic test for
said disease.
[0284] In another embodiment, the intervention is a therapy for
said disease, more preferably a preventative therapy for said
disease.
[0285] Preferably, the disease is lung cancer, more preferably the
disease is lung cancer and the protective and susceptibility
polymorphisms are selected from the group consisting of: [0286] the
-133 G/C polymorphism in the Interleukin-18 gene; [0287] the -1053
C/T polymorphism in the CYP 2E1 gene; [0288] the Arg197Gln
polymorphism in the NAT2 gene; [0289] the -511 G/A polymorphism in
the Interleukin 1B gene; [0290] the Ala 9 Thr polymorphism in the
Anti-chymotrypsin gene; [0291] the S allele polymorphism in the
Alpha1-antitrypsin gene; [0292] the -251 A/T polymorphism in the
Interleukin-8 gene; [0293] the Lys 751 gln polymorphism in the XPD
gene; [0294] the +760 G/C polymorphism in the SOD3 gene; [0295] the
Phe257Ser polymorphism in the REV gene; [0296] the Z alelle
polymorphism in the Alpha1-antitrypsin gene; [0297] the R19W A/G
polymorphism in the Cerberus 1 (Cer 1) gene; the Ser307Ser G/T
polymorphism in the XRCC4 gene; [0298] the K3326X A/T polymorphism
in the BRCA2 gene; [0299] the V433M A/G polymorphism in the
Integrin alpha-11 gene;
[0300] the E375G T/C polymorphism in the CAMKK1 gene; [0301] the
A/T c74delA polymorphism in the gene encoding cytochrome P450
polypeptide CYP3A43, [0302] the A/C (rs2279115) polymorphism in the
gene encoding B-cell CLL/lymphoma 2, [0303] the A/G at +3100 in the
3'UTR (rs2317676) polymorphism of the gene encoding Integrin beta
3, [0304] the -3714 G/T (rs6413429) polymorphism in the gene
encoding Dopamine transporter 1, [0305] the A/G (rs1139417)
polymorphism in the gene encoding Tumor necrosis factor receptor 1,
[0306] the C/Del (rs1799732) polymorphism in the gene encoding
Dopamine receptor D2, [0307] the C/T (rs763110) polymorphism in the
gene encoding Fas ligand, [0308] the C/T (rs5743836) polymorphism
in the gene encoding Toll-like receptor 9, [0309] the -81 C/T (rs
2273953) polymorphism in the 5' UTR of the gene encoding Tumor
protein P73,
[0310] or one or more polymorphisms in linkage disequilibrium with
one or more of said polymorphisms.
[0311] More preferably, said intervention is a CT scan for lung
cancer.
[0312] Still more preferably, the method is as described herein
with reference to the examples and/or figures.
[0313] In a further aspect, the present invention provides a kit
for assessing a subject's risk of developing lung cancer, said kit
comprising a means of analysing a sample from said subject for the
presence or absence of one or more polymorphisms disclosed
herein.
BRIEF DESCRIPTION OF FIGURES
[0314] FIG. 1: depicts a graph showing the likelihood of having
lung cancer plotted against the SNP score derived from the 5 SNP
panel shown in Table 16 herein.
[0315] FIG. 2: depicts a graph showing the log odds of having lung
cancer plotted against the SNP score derived from the 5 SNP panel
shown in Table 16 herein.
[0316] FIG. 3 depicts a graph showing the likelihood of having lung
cancer plotted against the SNP score derived from an 11 SNP panel
(11 SNP panel A) comprising SNPs 1-11 in Table 18 herein.
[0317] FIG. 4 depicts a receiver-operator curve analysis of
sensitivity and specificity for the 11 SNP panel A.
[0318] FIG. 5 depicts a graph showing the distribution of
frequencies of control smokers and lung cancer subjects plotted
against SNP score derived from the 11 SNP panel A.
[0319] FIG. 6 depicts a graph showing the likelihood of having lung
cancer plotted against the SNP score derived from a 16 SNP panel
comprising SNPs 1-16 in Table 18 herein.
[0320] FIG. 7 depicts a receiver-operator curve analysis of
sensitivity and specificity for the 16 SNP panel.
[0321] FIG. 8 depicts a graph showing the distribution of
frequencies of control smokers and lung cancer subjects plotted
against SNP score derived from the 16 SNP panel.
[0322] FIG. 9 depicts a graph showing the log odds of having lung
cancer plotted against the SNP score derived from the 9 SNP panel
described herein.
[0323] FIG. 10 depicts a receiver-operator curve analysis of
sensitivity and specificity for the 9 SNP panel.
[0324] FIG. 11 depicts a graph showing the distribution of
frequencies of control smokers and lung cancer subjects plotted
against SNP score derived from the 9 SNP panel.
[0325] FIG. 12 depicts a graph showing the likelihood of having one
of the four common types of lung cancer plotted against the SNP
score, as described in Example 5.
[0326] FIG. 13a depicts a graph showing the frequency of lung
cancer plotted against the SNP score derived from the 19 SNP panel
described in Example 6 herein.
[0327] FIG. 13b depicts a graph showing the odds ratio of lung
cancer according to the SNP score derived from the 19 SNP panel
described in Example 6 herein.
[0328] FIG. 14 depicts a graph showing the distribution of
frequencies of control smokers and lung cancer subjects plotted
against SNP score derived from the 19 SNP panel described in
Example 6 herein.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0329] Using case-control studies the frequencies of several
genetic variants (polymorphisms) of candidate genes in smokers who
have developed lung cancer and blood donor controls have been
compared. The majority of these candidate genes have confirmed (or
likely) functional effects on gene expression or protein function.
Specifically the frequencies of polymorphisms between blood donor
controls, resistant smokers and those with lung cancer (subdivided
into those with early onset and those with normal onset) have been
compared. The present invention demonstrates that there are both
protective and susceptibility polymorphisms present in selected
candidate genes of the patients tested.
[0330] In one embodiment described herein 8 susceptibility genetic
polymorphisms and 6 protective genetic polymorphism are identified.
These are as follows:
TABLE-US-00001 Gene and SNP rs number Genotype Phenotype OR P value
Cerberus 1 (Cer 1) R19W A/G rs10115703 AA/AG susceptiblility 1.7
0.02 XRCC4 Ser307Ser G/T rs1056503 GG/GT susceptiblility 1.3 0.04
BRCA2 K3326X A/T rs11571833 AT/TT susceptiblility 2.5 0.04 Integrin
alpha-11 V433M A/G rs2306022 AA susceptiblility 4.3 0.002 CAMKK1
E375G T/C rs7214723 TT protective 0.76 0.13 P73 rs2273953 CC
protective 0.46 <0.001 CYP3A43 C74 delA AT/TT susceptiblility
1.74 0.05 BCL2 rs2279115 AA protective 0.69 0.05 ITGB3 rs2317676
AG/GG protective 0.57 0.02 DAT1 rs6413429 GT/TT susceptibility 1.6
0.05 TNFR1 rs1139417 AA susceptibility 1.5 0.02 DRD2 rs1799732
CDel/DelDel protective 0.61 0.02 FasL rs763110 TT protective 0.61
0.05 TLR9 rs5743836 CC susceptibility 3.1 0.03
[0331] A susceptibility genetic polymorphism is one which, when
present, is indicative of an increased risk of developing lung
cancer. In contrast, a protective genetic polymorphism is one
which, when present, is indicative of a reduced risk of developing
lung cancer.
[0332] As used herein, the phrase "risk of developing lung cancer"
means the likelihood that a subject to whom the risk applies will
develop lung cancer, and includes predisposition to, and potential
onset of the disease. Accordingly, the phrase "increased risk of
developing lung cancer" means that a subject having such an
increased risk possesses an hereditary inclination or tendency to
develop lung cancer. This does not mean that such a person will
actually develop lung cancer at any time, merely that he or she has
a greater likelihood of developing lung cancer compared to the
general population of individuals that either does not possess a
polymorphism associated with increased lung cancer or does possess
a polymorphism associated with decreased lung cancer risk. Subjects
with an increased risk of developing lung cancer include those with
a predisposition to lung cancer, such as a tendency or predilection
regardless of their lung function at the time of assessment, for
example, a subject who is genetically inclined to lung cancer but
who has normal lung function, those at potential risk, including
subjects with a tendency to mildly reduced lung function who are
likely to go on to suffer lung cancer if they keep smoking, and
subjects with potential onset of lung cancer, who have a tendency
to poor lung function on spirometry etc., consistent with lung
cancer at the time of assessment.
[0333] Similarly, the phrase "decreased risk of developing lung
cancer" means that a subject having such a decreased risk possesses
an hereditary disinclination or reduced tendency to develop lung
cancer. This does not mean that such a person will not develop lung
cancer at any time, merely that he or she has a decreased
likelihood of developing lung cancer compared to the general
population of individuals that either does possess one or more
polymorphisms associated with increased lung cancer, or does not
possess a polymorphism associated with decreased lung cancer.
[0334] It will be understood that in the context of the present
invention the term "polymorphism" means the occurrence together in
the same population at a rate greater than that attributable to
random mutation (usually greater than 1%) of two or more alternate
forms (such as alleles or genetic markers) of a chromosomal locus
that differ in nucleotide sequence or have variable numbers of
repeated nucleotide units. See
www.ornl.gov/sci/techresources/Human_Genome/publicat/97pr/09gloss.html#p.
Accordingly, the term "polymorphisms" is used herein contemplates
genetic variations, including single nucleotide substitutions,
insertions and deletions of nucleotides, repetitive sequences (such
as microsatellites), and the total or partial absence of genes (eg.
null mutations). As used herein, the term "polymorphisms" also
includes genotypes and haplotypes. A genotype is the genetic
composition at a specific locus or set of loci. A haplotype is a
set of closely linked genetic markers present on one chromosome
which are not easily separable by recombination, tend to be
inherited together, and may be in linkage disequilibrium. A
haplotype can be identified by patterns of polymorphisms such as
SNPs. Similarly, the term "single nucleotide polymorphism" or "SNP"
in the context of the present invention includes single base
nucleotide substitutions and short deletion and insertion
polymorphisms.
[0335] A reduced or increased risk of a subject developing lung
cancer may be diagnosed by analysing a sample from said subject for
the presence of a polymorphism selected from the group consisting
of: [0336] R19W A/G (rs10115703) in the gene encoding Cerberus 1
(Cer 1); [0337] Ser307Ser G/T (rs1056503) in the X-ray repair
complementing defective repair in Chinese hamster cells 4 gene
(XRCC4); [0338] K3326X A/T (rs 1571833) in the breast cancer 2
early onset gene (BRCA2); [0339] V433M A/G (rs2306022) in the gene
encoding Integrin alpha-1; [0340] E375G T/C (rs7214723) in the gene
encoding Calcium/calmodulin-dependent protein kinase kinase 1
(CAMKK1); [0341] A/T c74delA in the gene encoding cytochrome P450
polypeptide CYP3A43 (CYP3A43); [0342] A/C (rs2279115) in the gene
encoding B-cell CLL/lymphoma 2 (BCL2); [0343] A/G at +3100 in the
3'UTR (rs2317676) of the gene encoding Integrin beta 3 (ITGB3);
[0344] G/T (rs6413429) in the gene encoding Dopamine transporter 1
(DAT1); A/G (rs1139417) in the gene encoding Tumor necrosis factor
receptor 1 (TNFR1); [0345] C/Del (rs1799732) in the gene encoding
Dopamine receptor D2 (DRD2); [0346] C/T (rs763110) in the gene
encoding Fas ligand (FasL); or [0347] C/T (rs5743836) in the gene
encoding Toll-like receptor 9 (TLR9) [0348] -81 C/T (rs 2273953) in
the 5' UTR of the gene encoding Tumor protein P73 (P73); or one or
more polymorphisms which are in linkage disequilibrium with any one
or more of the above group.
[0349] These polymorphisms can also be analysed in combinations of
two or more, or in combination with other polymorphisms indicative
of a subject's risk of developing lung cancer inclusive of the
remaining polymorphisms listed above.
[0350] Expressly contemplated are combinations of the above
polymorphisms with polymorphisms as described in PCT International
application PCT/NZ02/00106, published as WO 02/099134, or as
described in PCT International application PCT/NZ2006/000125,
published as WO2006/123955, or those polymorphisms recited herein
in Table 18.
[0351] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0352] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0353] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0354] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0355] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0356] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0357] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0358] Arg 312 Gln (rs 1799895) in the gene encoding SOD3;
[0359] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0360] C/Del (rs1799732) in the gene encoding DRD2;
[0361] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0362] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0363] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0364] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0365] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0366] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0367] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0368] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0369] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0370] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0371] C/Del (rs1799732) in the gene encoding DRD2;
[0372] A/C (rs2279115) in the gene encoding BCL2;
[0373] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0374] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected:
[0375] -133 G/C (rs360721) in the promoter of the gene encoding
Interleukin-18;
[0376] -251 A/T (rs4073) in the gene encoding Interleukin-8;
[0377] Arg 197 Gln (rs 1799930) in the gene encoding
N-acetylcysteine transferase 2;
[0378] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin;
[0379] -3714 G/T (rs6413429) in the gene encoding DAT 1;
[0380] -81 C/T (rs 2273953) in the 5' UTR of the gene encoding
P73;
[0381] Arg 312 Gln (rs1799895) in the gene encoding SOD3;
[0382] A/G at +3100 in the 3'UTR (rs2317676) of the gene encoding
ITGB3;
[0383] C/Del (rs1799732) in the gene encoding DRD2;
[0384] A/C (rs2279115) in the gene encoding BCL2;
[0385] V433M A/G (rs2306022) in the gene encoding ITGA11;
[0386] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0387] In one embodiment of the methods and uses of the present
invention each of the following polymorphisms are selected: [0388]
Rsa 1 C/T (rs2031920) in the gene encoding CYP 2E1; [0389] -133 G/C
(rs360721) in the promoter of the gene encoding Interleukin-18;
[0390] -251 A/T (rs4073) in the gene encoding Interleukin-8; [0391]
-511 A/G (rs 16944) in the gene encoding Interleukin 1B; [0392]
V433M A/G (rs2306022) in the gene encoding ITGA11; [0393] Arg 197
Gln A/G (rs 1799930) in the gene encoding N-acetylcysteine
transferase 2; [0394] Ala 15 Thr A/G (rs4934) in the gene encoding
.alpha.1-antichymotrypsin; [0395] R19W A/G in the gene encoding
Cerberus 1 (rs 10115703); [0396] -3714 G/T (rs6413429) in the gene
encoding DAT1 (rs6413429); [0397] A/G (rs1139417) in the gene
encoding TNF.alpha.1; [0398] C/T (rs5743836) in the gene encoding
TLR9; [0399] -81 C/T (rs 2273953) in the 5' UTR of the gene
encoding P73; [0400] Arg 312 Gln (rs1799895) in the gene encoding
SOD3; [0401] A/G at +3100 in the 3' UTR (rs2317676) of the gene
encoding ITGB3; [0402] C/Del (rs1799732) in the gene encoding DRD2;
[0403] A/C (rs2279115) in the gene encoding BCL2; [0404] -751 G/T
(rs 13181) in the promoter of the gene encoding XPD; [0405] Phe 257
Ser C/T (rs3087386) in the gene encoding REV1; [0406] C/T
(rs763110) in the gene encoding FasL;
[0407] or one or more polymorphisms in linkage disequilibrium with
any one or more of these polymorphisms.
[0408] Assays which involve combinations of polymorphisms,
including those amenable to high throughput, such as those
utilising microarrays, are preferred.
[0409] Statistical analyses, particularly of the combined effects
of these polymorphisms, show that the genetic analyses of the
present invention can be used to determine the risk quotient of any
smoker and in particular to identify smokers at greater risk of
developing lung cancer. Such combined analysis can be of
combinations of susceptibility polymorphisms only, of protective
polymorphisms only, or of combinations of both. Analysis can also
be step-wise, with analysis of the presence or absence of
protective polymorphisms occurring first and then with analysis of
susceptibility polymorphisms proceeding only where no protective
polymorphisms are present.
[0410] Thus, through systematic analysis of the frequency of these
polymorphisms in well defined groups of smokers and non-smokers, as
described herein, it is possible to implicate certain proteins in
the development of lung cancer and improve the ability to identify
which smokers are at increased risk of developing lung
cancer-related impaired lung function and lung cancer for
predictive purposes.
[0411] The present results show for the first time that the
minority of smokers who develop lung cancer do so because they have
one or more of the susceptibility polymorphisms and few or none of
the protective polymorphisms defined herein. It is thought that the
presence of one or more suscetptible polymorphisms, together with
the damaging irritant and oxidant effects of smoking, combine to
make this group of smokers highly susceptible to developing lung
cancer. Additional risk factors, such as familial history, age,
weight, pack years, etc., will also have an impact on the risk
profile of a subject, and can be assessed in combination with the
genetic analyses described herein.
[0412] The one or more polymorphisms can be detected directly or by
detection of one or more polymorphisms which are in linkage
disequilibrium with said one or more polymorphisms. As discussed
above, linkage disequilibrium is a phenomenon in genetics whereby
two or more mutations or polymorphisms are in such close genetic
proximity that they are co-inherited. This means that in
genotyping, detection of one polymorphism as present infers the
presence of the other. (Reich D E et al; Linkage disequilibrium in
the human genome, Nature 2001, 411: 199-204.)
[0413] It will be apparent that polymorphsisms in linkage
disequilibrium with one or more other polymorphism associated with
increased or decreased risk of developing lung cancer will also
provide utility as biomarkers for risk of developing lung cancer.
The data presented herein shows that the frequency for SNPs in
linkage disequilibrium is very similar. Accordingly, these
genetically linked SNPs can be utilized in combined polymorphism
analyses to derive a level of risk comparable to that calculated
from the original SNP.
[0414] It will therefore be apparent that one or more polymorphisms
in linkage disequilibrium with the polymorphisms specified herein
can be identified, for example, using public data bases. Examples
of such polymorphisms reported to be in linkage disequilibrium with
the polymorphisms specified herein are presented herein in Table
26.
[0415] It will also be apparent that frequently a variety of
nomenclatures may exist for any given polymorphism or for any given
gene. For example, the polymorphism Arg 312 Gln in the gene
encoding superoxide dismutase 3 (SOD3) is believed to have been
referred to variously as Arg 213 Gly, +760 G/C, and Arg 231 Gly (rs
1799895). In another example, the gene referred to herein as the
breast cancer 2 early onset gene is also variously referred to as
BRCC2, Breast Cancer 2 Gene, Breast Cancer Type 2, Breast Cancer
Type 2 Susceptibility Gene, Breast cancer type 2 susceptibility
protein, FACD, FAD, FAD 1, FANCB, FANCD 1, and Hereditary Breast
Cancer 2. When referring to a susceptibility or protective
polymorphism as herein described, such alternative nomenclatures
are also contemplated by the present invention.
[0416] The methods of the invention are primarily directed to the
detection and identification of the above polymorphisms associated
with lung cancer, which are all single nucleotide polymorphisms. In
general terms, a single nucleotide polymorphism (SNP) is a single
base change or point mutation resulting in genetic variation
between individuals. SNPs occur in the human genome approximately
once every 100 to 300 bases, and can occur in coding or non-coding
regions. Due to the redundancy of the genetic code, a SNP in the
coding region may or may not change the amino acid sequence of a
protein product. A SNP in a non-coding region can, for example,
alter gene expression by, for example, modifying control regions
such as promoters, transcription factor binding sites, processing
sites, ribosomal binding sites, and affect gene transcription,
processing, and translation.
[0417] SNPs can facilitate large-scale association genetics
studies, and there has recently been great interest in SNP
discovery and detection. SNPs show great promise as markers for a
number of phenotypic traits (including latent traits), such as for
example, disease propensity and severity, wellness propensity, and
drug responsiveness including, for example, susceptibility to
adverse drug reactions. Knowledge of the association of a
particular SNP with a phenotypic trait, coupled with the knowledge
of whether an individual has said particular SNP, can enable the
targeting of diagnostic, preventative and therapeutic applications
to allow better disease management, to enhance understanding of
disease states and to ultimately facilitate the discovery of more
effective treatments, such as personalised treatment regimens.
[0418] Indeed, a number of databases have been constructed of known
SNPs, and for some such SNPs, the biological effect associated with
a SNP. For example, the NCBI SNP database "dbSNP" is incorporated
into NCBI's Entrez system and can be queried using the same
approach as the other Entrez databases such as PubMed and GenBank.
This database has records for over 1.5 million SNPs mapped onto the
human genome sequence. Each dbSNP entry includes the sequence
context of the polymorphism (i.e., the surrounding sequence), the
occurrence frequency of the polymorphism (by population or
individual), and the experimental method(s), protocols, and
conditions used to assay the variation, and can include information
associating a SNP with a particular phenotypic trait.
[0419] At least in part because of the potential impact on health
and wellness, there has been and continues to be a great deal of
effort to develop methods that reliably and rapidly identify SNPs.
Initially, this was no trivial task, at least in part because of
the complexity of human genomic DNA, with a haploid genome of
3.times.10.sup.9 base pairs, and the associated sensitivity and
discriminatory requirements.
[0420] Genotyping approaches to detect SNPs well-known in the art
include DNA sequencing, methods that require allele specific
hybridization of primers or probes, allele specific incorporation
of nucleotides to primers bound close to or adjacent to the
polymorphisms (often referred to as "single base extension", or
"minisequencing"), allele-specific ligation (oining) of
oligonucleotides (ligation chain reaction or ligation padlock
probes), allele-specific cleavage of oligonucleotides or PCR
products by restriction enzymes (restriction fragment length
polymorphisms analysis or RFLP) or chemical or other agents,
resolution of allele-dependent differences in electrophoretic or
chromatographic mobilities, by structure specific enzymes including
invasive structure specific enzymes, or mass spectrometry. Analysis
of amino acid variation is also possible where the SNP lies in a
coding region and results in an amino acid change.
[0421] DNA sequencing allows the direct determination and
identification of SNPs. The benefits in specificity and accuracy
are generally outweighed for screening purposes by the difficulties
inherent in whole genome, or even targeted subgenome,
sequencing.
[0422] Mini-sequencing involves allowing a primer to hybridize to
the DNA sequence adjacent to the SNP site on the test sample under
investigation. The primer is extended by one nucleotide using all
four differentially tagged fluorescent dideoxynucleotides (A, C, G,
or T), and a DNA polymerase. Only one of the four nucleotides
(homozygous case) or two of the four nucleotides (heterozygous
case) is incorporated. The base that is incorporated is
complementary to the nucleotide at the SNP position.
[0423] A number of methods currently used for SNP detection involve
site-specific and/or allele-specific hybridisation. These methods
are largely reliant on the discriminatory binding of
oligonucleotides to target sequences containing the SNP of
interest. The techniques of Affymetrix (Santa Clara, Calif.) and
Nanogen Inc. (San Diego, Calif.) are particularly well-known, and
utilize the fact that DNA duplexes containing single base
mismatches are much less stable than duplexes that are perfectly
base-paired. The presence of a matched duplex is detected by
fluorescence.
[0424] The majority of methods to detect or identify SNPs by
site-specific hybridisation require target amplification by methods
such as PCR to increase sensitivity and specificity (see, for
example U.S. Pat. No. 5,679,524, PCT publication WO 98/59066, PCT
publication WO 95/12607). US Application 20050059030 (incorporated
herein in its entirety) describes a method for detecting a single
nucleotide polymorphism in total human DNA without prior
amplification or complexity reduction to selectively enrich for the
target sequence, and without the aid of any enzymatic reaction. The
method utilises a single-step hybridization involving two
hybridization events: hybridization of a first portion of the
target sequence to a capture probe, and hybridization of a second
portion of said target sequence to a detection probe. Both
hybridization events happen in the same reaction, and the order in
which hybridisation occurs is not critical.
[0425] US Application 20050042608 (incorporated herein in its
entirety) describes a modification of the method of electrochemical
detection of nucleic acid hybridization of Thorp et al. (U.S. Pat.
No. 5,871,918). Briefly, capture probes are designed, each of which
has a different SNP base and a sequence of probe bases on each side
of the SNP base. The probe bases are complementary to the
corresponding target sequence adjacent to the SNP site. Each
capture probe is immobilized on a different electrode having a
non-conductive outer layer on a conductive working surface of a
substrate. The extent of hybridization between each capture probe
and the nucleic acid target is detected by detecting the
oxidation-reduction reaction at each electrode, utilizing a
transition metal complex. These differences in the oxidation rates
at the different electrodes are used to determine whether the
selected nucleic acid target has a single nucleotide polymorphism
at the selected SNP site.
[0426] The technique of Lynx Therapeutics (Hayward, Calif.) using
MEGATYPE.TM. technology can genotype very large numbers of SNPs
simultaneously from small or large pools of genomic material. This
technology uses fluorescently labeled probes and compares the
collected genomes of two populations, enabling detection and
recovery of DNA fragments spanning SNPs that distinguish the two
populations, without requiring prior SNP mapping or knowledge.
[0427] A number of other methods for detecting and identifying SNPs
exist. These include the use of mass spectrometry, for example, to
measure probes that hybridize to the SNP. This technique varies in
how rapidly it can be performed, from a few samples per day to a
high throughput of 40,000 SNPs per day, using mass code tags. A
preferred example is the use of mass spectrometric determination of
a nucleic acid sequence which comprises the polymorphisms of the
invention, for example, as shown herein in the Examples. Such mass
spectrometric methods are known to those skilled in the art, and
the genotyping methods of the invention are amenable to adaptation
for the mass spectrometric detection of the polymorphisms of the
invention, for example, the polymorphisms of the invention as shown
in Table 16 herein.
[0428] SNPs can also be determined by ligation-bit analysis. This
analysis requires two primers that hybridize to a target with a one
nucleotide gap between the primers. Each of the four nucleotides is
added to a separate reaction mixture containing DNA polymerase,
ligase, target DNA and the primers. The polymerase adds a
nucleotide to the 3' end of the first primer that is complementary
to the SNP, and the ligase then ligates the two adjacent primers
together. Upon heating of the sample, if ligation has occurred, the
now larger primer will remain hybridized and a signal, for example,
fluorescence, can be detected. A further discussion of these
methods can be found in U.S. Pat. Nos. 5,919,626; 5,945,283;
5,242,794; and 5,952,174.
[0429] U.S. Pat. No. 6,821,733 (incorporated herein in its
entirety) describes methods to detect differences in the sequence
of two nucleic acid molecules that includes the steps of:
contacting two nucleic acids under conditions that allow the
formation of a four-way complex and branch migration; contacting
the four-way complex with a tracer molecule and a detection
molecule under conditions in which the detection molecule is
capable of binding the tracer molecule or the four-way complex; and
determining binding of the tracer molecule to the detection
molecule before and after exposure to the four-way complex.
Competition of the four-way complex with the tracer molecule for
binding to the detection molecule indicates a difference between
the two nucleic acids.
[0430] Protein- and proteomics-based approaches are also suitable
for polymorphism detection and analysis. Polymorphisms which result
in or are associated with variation in expressed proteins can be
detected directly by analysing said proteins. This typically
requires separation of the various proteins within a sample, by,
for example, gel electrophoresis or HPLC, and identification of
said proteins or peptides derived therefrom, for example by NMR or
protein sequencing such as chemical sequencing or more prevalently
mass spectrometry. Proteomic methodologies are well known in the
art, and have great potential for automation. For example,
integrated systems, such as the ProteomIQ.TM. system from Proteome
Systems, provide high throughput platforms for proteome analysis
combining sample preparation, protein separation, image acquisition
and analysis, protein processing, mass spectrometry and
bioinformatics technologies.
[0431] The majority of proteomic methods of protein identification
utilise mass spectrometry, including ion trap mass spectrometry,
liquid chromatography (LC) and LC/MSn mass spectrometry, gas
chromatography (GC) mass spectroscopy, Fourier transform-ion
cyclotron resonance-mass spectrometer (FT-MS), MALDI-TOF mass
spectrometry, and ESI mass spectrometry, and their derivatives.
Mass spectrometric methods are also useful in the determination of
post-translational modification of proteins, such as
phosphorylation or glycosylation, and thus have utility in
determining polymorphisms that result in or are associated with
variation in post-translational modifications of proteins.
[0432] Associated technologies are also well known, and include,
for example, protein processing devices such as the "Chemical
Inkjet Printer" comprising piezoelectric printing technology that
allows in situ enzymatic or chemical digestion of protein samples
electroblotted from 2-D PAGE gels to membranes by jetting the
enzyme or chemical directly onto the selected protein spots. After
in-situ digestion and incubation of the proteins, the membrane can
be placed directly into the mass spectrometer for peptide
analysis.
[0433] A large number of methods reliant on the conformational
variability of nucleic acids have been developed to detect
SNPs.
[0434] For example, Single Strand Conformational Polymorphism
(SSCP, Orita et al., PNAS 198986:2766-2770) is a method reliant on
the ability of single-stranded nucleic acids to form secondary
structure in solution under certain conditions. The secondary
structure depends on the base composition and can be altered by a
single nucleotide substitution, causing differences in
electrophoretic mobility under nondenaturing conditions. The
various polymorphs are typically detected by autoradiography when
radioactively labelled, by silver staining of bands, by
hybridisation with detectably labelled probe fragments or the use
of fluorescent PCR primers which are subsequently detected, for
example by an automated DNA sequencer.
[0435] Modifications of SSCP are well known in the art, and include
the use of differing gel running conditions, such as for example
differing temperature, or the addition of additives, and different
gel matrices. Other variations on SSCP are well known to the
skilled artisan, including, RNA-SSCP, restriction endonuclease
fingerprinting-SSCP, dideoxy fingerprinting (a hybrid between
dideoxy sequencing and SSCP), bi-directional dideoxy fingerprinting
(in which the dideoxy termination reaction is performed
simultaneously with two opposing primers), and Fluorescent PCR-SSCP
(in which PCR products are internally labelled with multiple
fluorescent dyes, may be digested with restriction enzymes,
followed by SSCP, and analysed on an automated DNA sequencer able
to detect the fluorescent dyes).
[0436] Other methods which utilise the varying mobility of
different nucleic acid structures include Denaturing Gradient Gel
Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis
(TGGE), and Heteroduplex Analysis (HET). Here, variation in the
dissociation of double stranded DNA (for example, due to base-pair
mismatches) results in a change in electrophoretic mobility. These
mobility shifts are used to detect nucleotide variations.
[0437] Denaturing High Pressure Liquid Chromatography (HPLC) is yet
a further method utilised to detect SNPs, using HPLC methods
well-known in the art as an alternative to the separation methods
described above (such as gel electophoresis) to detect, for
example, homoduplexes and heteroduplexes which elute from the HPLC
column at different rates, thereby enabling detection of mismatch
nucleotides and thus SNPs.
[0438] Yet further methods to detect SNPs rely on the differing
susceptibility of single stranded and double stranded nucleic acids
to cleavage by various agents, including chemical cleavage agents
and nucleolytic enzymes. For example, cleavage of mismatches within
RNA:DNA heteroduplexes by RNase A, of heteroduplexes by, for
example bacteriophage T4 endonuclease YII or T7 endonuclease I, of
the 5' end of the hairpin loops at the junction between single
stranded and double stranded DNA by cleavase I, and the
modification of mispaired nucleotides within heteroduplexes by
chemical agents commonly used in Maxam-Gilbert sequencing
chemistry, are all well known in the art.
[0439] Further examples include the Protein Translation Test (PTT),
used to resolve stop codons generated by variations which lead to a
premature termination of translation and to protein products of
reduced size, and the use of mismatch binding proteins. Variations
are detected by binding of, for example, the MutS protein, a
component of Escherichia coli DNA mismatch repair system, or the
human hMSH2 and GTBP proteins, to double stranded DNA
heteroduplexes containing mismatched bases. DNA duplexes are then
incubated with the mismatch binding protein, and variations are
detected by mobility shift assay. For example, a simple assay is
based on the fact that the binding of the mismatch binding protein
to the heteroduplex protects the heteroduplex from exonuclease
degradation.
[0440] Those skilled in the art will know that a particular SNP,
particularly when it occurs in a regulatory region of a gene such
as a promoter, can be associated with altered expression of a gene.
Altered expression of a gene can also result when the SNP is
located in the coding region of a protein-encoding gene, for
example where the SNP is associated with codons of varying usage
and thus with tRNAs of differing abundance. Such altered expression
can be determined by methods well known in the art, and can thereby
be employed to detect such SNPs. Similarly, where a SNP occurs in
the coding region of a gene and results in a non-synonomous amino
acid substitution, such substitution can result in a change in the
function of the gene product. Similarly, in cases where the gene
product is an RNA, such SNPs can result in a change of function in
the RNA gene product. Any such change in function, for example as
assessed in an activity or functionality assay, can be employed to
detect such SNPs.
[0441] The above methods of detecting and identifying SNPs are
amenable to use in the methods of the invention.
[0442] Of course, in order to detect and identify SNPs in
accordance with the invention, a sample containing material to be
tested is obtained from the subject. The sample can be any sample
potentially containing the target SNPs (or target polypeptides, as
the case may be) and obtained from any bodily fluid (blood, urine,
saliva, etc) biopsies or other tissue preparations.
[0443] DNA or RNA can be isolated from the sample according to any
of a number of methods well known in the art. For example, methods
of purification of nucleic acids are described in Tijssen;
Laboratory Techniques in Biochemistry and Molecular Biology:
Hybridization with nucleic acid probes Part 1: Theory and Nucleic
acid preparation, Elsevier, New York, N.Y. 1993, as well as in
Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning
Manual 1989.
[0444] To assist with detecting the presence or absence of
polymorphisms/SNPs, nucleic acid probes and/or primers can be
provided. Such probes have nucleic acid sequences specific for
chromosomal changes evidencing the presence or absence of the
polymorphism and are preferably labeled with a substance that emits
a detectable signal when combined with the target polymorphism.
[0445] The nucleic acid probes can be genomic DNA or cDNA or mRNA,
or any RNA-like or DNA-like material, such as peptide nucleic
acids, branched DNAs, and the like. The probes can be sense or
antisense polynucleotide probes. Where target polynucleotides are
double-stranded, the probes may be either sense or antisense
strands. Where the target polynucleotides are single-stranded, the
probes are complementary single strands.
[0446] The probes can be prepared by a variety of synthetic or
enzymatic schemes, which are well known in the art. The probes can
be synthesized, in whole or in part, using chemical methods well
known in the art (Caruthers et al., Nucleic Acids Res., Symp. Ser.,
215-233 (1980)). Alternatively, the probes can be generated, in
whole or in part, enzymatically.
[0447] Nucleotide analogs can be incorporated into probes by
methods well known in the art. The only requirement is that the
incorporated nucleotide analog must serve to base pair with target
polynucleotide sequences. For example, certain guanine nucleotides
can be substituted with hypoxanthine, which base pairs with
cytosine residues. However, these base pairs are less stable than
those between guanine and cytosine. Alternatively, adenine
nucleotides can be substituted with 2,6-diaminopurine, which can
form stronger base pairs than those between adenine and
thymidine.
[0448] Additionally, the probes can include nucleotides that have
been derivatized chemically or enzymatically. Typical chemical
modifications include derivatization with acyl, alkyl, aryl or
amino groups.
[0449] The probes can be immobilized on a substrate. Preferred
substrates are any suitable rigid or semi-rigid support including
membranes, filters, chips, slides, wafers, fibers, magnetic or
nonmagnetic beads, gels, tubing, plates, polymers, microparticles
and capillaries. The substrate can have a variety of surface forms,
such as wells, trenches, pins, channels and pores, to which the
polynucleotide probes are bound. Preferably, the substrates are
optically transparent.
[0450] Furthermore, the probes do not have to be directly bound to
the substrate, but rather can be bound to the substrate through a
linker group. The linker groups are typically about 6 to 50 atoms
long to provide exposure to the attached probe. Preferred linker
groups include ethylene glycol oligomers, diamines, diacids and the
like. Reactive groups on the substrate surface react with one of
the terminal portions of the linker to bind the linker to the
substrate. The other terminal portion of the linker is then
functionalized for binding the probe.
[0451] The probes can be attached to a substrate by dispensing
reagents for probe synthesis on the substrate surface or by
dispensing preformed DNA fragments or clones on the substrate
surface. Typical dispensers include a micropipette delivering
solution to the substrate with a robotic system to control the
position of the micropipette with respect to the substrate. There
can be a multiplicity of dispensers so that reagents can be
delivered to the reaction regions simultaneously.
[0452] Nucleic acid microarrays are preferred. Such microarrays
(including nucleic acid chips) are well known in the art (see, for
example U.S. Pat. Nos. 5,578,832; 5,861,242; 6,183,698; 6,287,850;
6,291,183; 6,297,018; 6,306,643; and 6,308,170, each incorporated
by reference).
[0453] Alternatively, antibody microarrays can be produced. The
production of such microarrays is essentially as described in
Schweitzer & Kingsmore, "Measuring proteins on microarrays",
Curr Opin Biotechnol 2002; 13(1): 14-9; Avseekno et al.,
"Immobilization of proteins in immunochemical microarrays
fabricated by electrospray deposition", Anal Chem 2001 15; 73(24):
6047-52; Huang, "Detection of multiple proteins in an
antibody-based protein microarray system, Immunol Methods 2001 1;
255 (1-2): 1-13.
[0454] The present invention also contemplates the preparation of
kits for use in accordance with the present invention. Suitable
kits include various reagents for use in accordance with the
present invention in suitable containers and packaging materials,
including tubes, vials, and shrink-wrapped and blow-molded
packages.
[0455] Materials suitable for inclusion in an exemplary kit in
accordance with the present invention comprise one or more of the
following: gene specific PCR primer pairs (oligonucleotides) that
anneal to DNA or cDNA sequence domains that flank the genetic
polymorphisms of interest, reagents capable of amplifying a
specific sequence domain in either genomic DNA or cDNA without the
requirement of performing PCR; reagents required to discriminate
between the various possible alleles in the sequence domains
amplified by PCR or non-PCR amplification (e.g., restriction
endonucleases, oligonucleotide that anneal preferentially to one
allele of the polymorphism, including those modified to contain
enzymes or fluorescent chemical groups that amplify the signal from
the oligonucleotide and make discrimination of alleles more
robust); reagents required to physically separate products derived
from the various alleles (e.g. agarose or polyacrylamide and a
buffer to be used in electrophoresis, HPLC columns, SSCP gels,
formamide gels or a matrix support for MALDI-TOF).
[0456] It will be appreciated that the methods of the invention can
be performed in conjunction with an analysis of other risk factors
known to be associated with lung cancer. Such risk factors include
epidemiological risk factors associated with an increased risk of
developing lung cancer. Such risk factors include, but are not
limited to smoking and/or exposure to tobacco smoke, age, sex and
familial history. These risk factors can be used to augment an
analysis of one or more polymorphisms as herein described when
assessing a subject's risk of developing lung cancer.
[0457] It is recognised that individual SNPs may confer weak risk
of susceptibility or protection to a disease or phenotype of
interest. These modest effects from individual SNPs are typically
measured as odds ratios in the order of 1-3. The specific phenotype
of interest may be a disease, such as lung cancer, or an
intermediate phenotype based on a pathological, biochemical or
physiological abnormality (for example, impaired lung function). As
shown herein, when specific genotypes from individual SNPs are
assigned a numerical value reflecting their phenotypic effect (for
example, a positive value for susceptibility SNPs and a negative
value for protective SNPs), the combined effects of these SNPs can
be derived from an algorithm that calculates an overall score.
Again as shown herein in a case-control study design, this SNP
score is linearly related to the frequency of disease (or
likelihood of having disease)-see for example FIGS. 3 and 4.
[0458] The SNP score provides a means of comparing people with
different scores and their odds of having disease in a simple
dose-response relationship. In this analysis, the people with the
lowest SNP score are the referent group (Odds ratio=1) and those
with greater SNP scores have a correspondingly greater odds (or
likelihood) of having the disease--again in a linear fashion. The
Applicants believe, without wishing to be bound by any theory, that
the extent to which combining SNPs optimises these analyses is
dependent, at least in part, on the strength of the effect of each
SNP individually in a univariate analysis (independent effect)
and/or multivariate analysis (effect after adjustment for effects
of other SNPs or non-genetic factors) and the frequency of the
genotype from that SNP (how common the SNP is). However, the effect
of combining certain SNPs may also be in part related to the effect
that those SNPs have on certain pathophysiological pathways that
underlie the phenotype or disease of interest.
[0459] The Applicants have found that combining certain SNPs may
increase the accuracy of the determination of risk or likelihood of
disease in an unpredictable fashion. Specifically, when the
distribution of SNP scores for the cases and controls are plotted
according to their frequency, the ability to segment those with and
without disease (or risk of disease) can be improved according to
the specific combination of SNPs that are analysed. See, for
example, the distributions for the 11 SNP panel A (FIG. 6) and for
the 16 SNP panel (FIG. 8). It appears that this effect is not
solely dependent on the number of relevant SNPs that are analysed
in combination, nor the magnitude of their individual effects, nor
their frequencies in the cases or controls. It further appears that
the ability to improve this segmentation of the population into
high and low risk is not due to any specific ratio of
susceptibility or protective SNPs. The Applicants believe, without
wishing to be bound by any theory, that the greater separation of
the population in to high and low risk may at least partly be a
function of identifying SNPs that confer a susceptibility or
protective phenotype in important but independent
pathophysiological pathways.
[0460] This observation has clinical utility in helping to define a
threshold or cut-off level in the SNP score that will define a
subgroup of the population to undergo an intervention. Such an
intervention may be a diagnostic intervention, such as imaging
test, other screening or diagnostic test (eg biochemical or RNA
based test), or may be a therapeutic intervention, such as a
chemopreventive therapy (for example, cisplatin or etoposide for
small cell lung cancer), radiotherapy, or a preventive lifestyle
modification (stopping smoking for lung cancer). In defining this
clinical threshold, people can be prioritised to a particular
intervention in such a way to minimise costs or minimise risks of
that intervention (for example, the costs of image-based screening
or expensive preventive treatment or risk from drug side-effects or
risk from radiation exposure). In determining this threshold, one
might aim to maximise the ability of the test to detect the
majority of cases (maximise sensitivity) but also to minimise the
number of people at low risk that require, or may be are otherwise
eligible for, the intervention of interest.
[0461] Receiver-operator curve (ROC) analyses analyze the clinical
performance of a test by examining the relationship between
sensitivity and false positive rate (i.e., 1-specificity) for a
single variable in a given population. In an ROC analysis, the test
variable may be derived from combining several factors. Either way,
this type of analysis does not consider the frequency distribution
of the test variable (for example, the SNP score) in the population
and therefore the number of people who would need to be screened in
order to identify the majority of those at risk but minimise the
number who need to be screened or treated. The Applicants have
found that this frequency distribution plot may be dependent on the
particular combination of SNPs under consideration and it appears
it may not be predicted by the effect conferred by each SNP on its
own nor from its performance characteristics (sensitivity and
specificity) in an ROC analysis.
[0462] The data presented herein shows that determining a specific
combination of SNPs can enhance the ability to segment or subgroup
people into intervention and non-intervention groups in order to
better prioritise these interventions. Such an approach is useful
in identifying which smokers might be best prioritised for
interventions, such as CT screening for lung cancer. Such an
approach could also be used for initiating treatments or other
screening or diagnostic tests. As will be appreciated, this has
important cost implications to offering such interventions.
[0463] Accordingly, the present invention also provides a method of
assessing a subject's suitability for an intervention diagnostic of
or therapeutic for a disease, the method comprising:
[0464] a) providing a net score for said subject, wherein the net
score is or has been determined by: [0465] i) providing the result
of one or more genetic tests of a sample from the subject, and
analysing the result for the presence or absence of protective
polymorphisms and for the presence or absence of susceptibility
polymorphisms, wherein said protective and susceptibility
polymorphisms are associated with said disease, [0466] ii)
assigning a positive score for each protective polymorphism and a
negative score for each susceptibility polymorphism or vice versa;
[0467] iii) calculating a net score for said subject by
representing the balance between the combined value of the
protective polymorphisms and the combined value of the
susceptibility polymorphisms present in the subject sample; and
[0468] b) providing a distribution of net scores for disease
sufferers and non-sufferers wherein the net scores for disease
sufferers and non-sufferers are or have been determined in the same
manner as the net score determined for said subject;
[0469] c) determining whether the net score for said subject lies
within a threshold on said distribution separating individuals
deemed suitable for said intervention from those for whom said
intervention is deemed unsuitable;
[0470] wherein a net score within said threshold is indicative of
the subject's suitability for the intervention, and wherein a net
score outside the threshold is indicative of the subject's
unsuitability for the intervention.
[0471] The value assigned to each protective polymorphism may be
the same or may be different. The value assigned to each
susceptibility polymorphism may be the same or may be different,
with either each protective polymorphism having a negative value
and each susceptibility polymorphism having a positive value, or
vice versa.
[0472] The intervention may be a diagnostic test for the disease,
such as a blood test or a CT scan for lung cancer. Alternatively,
the intervention may be a therapy for the disease, such as
chemotherapy or radiotherapy, including a preventative therapy for
the disease, such as the provision of motivation to the subject to
stop smoking.
[0473] As described herein, a distribution of SNP scores for lung
cancer sufferers and resistant smoker controls (non-sufferers) can
be established using the methods of the invention. For example, a
distribution of SNP scores derived from the 16 SNP panel consisting
of the protective and susceptibility polymorphisms selected from
the group consisting of the -133 G/C polymorphism in the
Interleukin-18 gene, the -1053 C/T polymorphism in the CYP 2E1
gene, the Arg197gln polymorphism in the Nat2 gene, the -511 G/A
polymorphism in the Interleukin 1B gene, the Ala 9 Thr polymorphism
in the Anti-chymotrypsin gene, the S allele polymorphism in the
Alpha1-antitrypsin gene, the -251 A/T polymorphism in the
Interleukin-8 gene, the Lys 751 gln polymorphism in the XPD gene,
the +760 G/C polymorphism in the SOD3 gene, the Phe257Ser
polymorphism in the REV gene, the Z alelle polymorphism in the
Alpha1-antitrypsin gene, the R19W A/G polymorphism in the Cerberus
1 (Cer 1) gene, the Ser307Ser G/T polymorphism in the XRCC4 gene,
the K3326X A/T polymorphism in the BRCA2 gene, the V433M A/G
polymorphism in the Integrin alpha-11 gene, and the E375G T/C
polymorphism in the CAMKK1 gene, among lung cancer sufferers and
non-sufferers is described herein. As shown herein, a threshold SNP
score can be determined that separates people into intervention and
non-intervention groups, so as to better prioritise those
individuals suitable for such interventions.
[0474] The predictive methods of the invention allow a number of
therapeutic interventions and/or treatment regimens to be assessed
for suitability and implemented for a given subject. The simplest
of these can be the provision to the subject of motivation to
implement a lifestyle change, for example, where the subject is a
current smoker, the methods of the invention can provide motivation
to quit smoking.
[0475] The manner of therapeutic intervention or treatment will be
predicated by the nature of the polymorphism(s) and the biological
effect of said polymorphism(s). For example, where a susceptibility
polymorphism is associated with a change in the expression of a
gene, intervention or treatment is preferably directed to the
restoration of normal expression of said gene, by, for example,
administration of an agent capable of modulating the expression of
said gene. Where a polymorphism is associated with decreased
expression of a gene, therapy can involve administration of an
agent capable of increasing the expression of said gene, and
conversely, where a polymorphism is associated with increased
expression of a gene, therapy can involve administration of an
agent capable of decreasing the expression of said gene. Methods
useful for the modulation of gene expression are well known in the
art. For example, in situations where a polymorphism is associated
with upregulated expression of a gene, therapy utilising, for
example, RNAi or antisense methodologies can be implemented to
decrease the abundance of mRNA and so decrease the expression of
said gene. Alternatively, therapy can involve methods directed to,
for example, modulating the activity of the product of said gene,
thereby compensating for the abnormal expression of said gene.
[0476] Where a susceptibility polymorphism is associated with
decreased gene product function or decreased levels of expression
of a gene product, therapeutic intervention or treatment can
involve augmenting or replacing of said function, or supplementing
the amount of gene product within the subject for example, by
administration of said gene product or a functional analogue
thereof. For example, where a polymorphism is associated with
decreased enzyme function, therapy can involve administration of
active enzyme or an enzyme analogue to the subject. Similarly,
where a polymorphism is associated with increased gene product
function, therapeutic intervention or treatment can involve
reduction of said function, for example, by administration of an
inhibitor of said gene product or an agent capable of decreasing
the level of said gene product in the subject. For example, where a
SNP allele or genotype is associated with increased enzyme
function, therapy can involve administration of an enzyme inhibitor
to the subject.
[0477] Likewise, when a protective polymorphism is associated with
upregulation of a particular gene or expression of an enzyme or
other protein, therapies can be directed to mimic such upregulation
or expression in an individual lacking the resistive genotype,
and/or delivery of such enzyme or other protein to such individual
Further, when a protective polymorphism is associated with
downregulation of a particular gene, or with diminished or
eliminated expression of an enzyme or other protein, desirable
therapies can be directed to mimicking such conditions in an
individual that lacks the protective genotype.
[0478] The relationship between the various polymorphisms
identified above and the susceptibility (or otherwise) of a subject
to lung cancer also has application in the design and/or screening
of candidate therapeutics. This is particularly the case where the
association between a susceptibility or protective polymorphism is
manifested by either an upregulation or downregulation of
expression of a gene. In such instances, the effect of a candidate
therapeutic on such upregulation or downregulation is readily
detectable.
[0479] For example, in one embodiment existing human lung organ and
cell cultures are screened for polymorphisms as set forth above.
(For information on human lung organ and cell cultures, see, e.g.:
Bohinski et al. (1996) Molecular and Cellular Biolog 14:5671-5681;
Collettsolberg et al. (1996) Pediatric Research 39:504; Hermanns et
al. (2004) Laboratory Investigation 84:736-752; Hume et al. (1996)
In Vitro Cellular & Developmental Biology-Animal 32:24-29;
Leonardi et al. (1995) 38:352-355; Notingher et al. (2003)
Biopolymers (Biospectroscopy) 72:230-240; Ohga et al. (1996)
Biochemical and Biophysical Research Communications 228:391-396;
each of which is hereby incorporated by reference in its entirety.)
Cultures representing susceptibility and protective genotype groups
are selected, together with cultures which are putatively "normal"
in terms of the expression of a gene which is either upregulated or
downregulated where a protective polymorphism is present.
[0480] Samples of such cultures are exposed to a library of
candidate therapeutic compounds and screened for any or all of: (a)
downregulation of susceptibility genes that are normally
upregulated in susceptibility polymorphisms; (b) upregulation of
susceptibility genes that are normally downregulated in
susceptibility polymorphisms; (c) downregulation of protective
genes that are normally downregulated or not expressed (or null
forms are expressed) in protective polymorphisms; and (d)
upregulation of protective genes that are normally upregulated in
protective polymorphisms. Compounds are selected for their ability
to alter the regulation and/or action of susceptibility genes
and/or protective genes in a culture having a susceptibility
polymorphisms.
[0481] Similarly, where the polymorphism is one which when present
results in a physiologically active concentration of an expressed
gene product outside of the normal range for a subject (adjusted
for age and sex), and where there is an available prophylactic or
therapeutic approach to restoring levels of that expressed gene
product to within the normal range, individual subjects can be
screened to determine the likelihood of their benefiting from that
restorative approach. Such screening involves detecting the
presence or absence of the polymorphism in the subject by any of
the methods described herein, with those subjects in which the
polymorphism is present being identified as individuals likely to
benefit from treatment.
[0482] The methods of the invention are primarily directed at
assessing risk of developing lung cancer. Lung cancer can be
divided into two main types based on histology--non-small cell
(approximately 80% of lung cancer cases) and small-cell (roughly
20% of cases) lung cancer. This histological division also reflects
treatment strategies and prognosis.
[0483] The non-small cell lung cancers (NSCLC) are generally
considered collectively because their prognosis and management is
roughly identical. For non-small cell lung cancer, prognosis is
poor. The most common types of NSCLC are adenocarcinoma, which
accounts for 50% to 60% of NSCLC, squamous cell carcinoma, and
large cell carcinoma.
[0484] Adenocarcinoma typically originates near the gas-exchanging
surface of the lung. Most cases of the adenocarcinoma are
associated with smoking. However, adenocarcinoma is the most common
form of lung cancer among non-smokers. A subtype of adenocarcinoma,
the bronchioalveolar carcinoma, is more common in female
non-smokers.
[0485] Squamous cell carcinoma, accounting for 20% to 25% of NSCLC,
generally originates in the larger breathing tubes. This is a
slower growing form of NSCLC.
[0486] Large cell carcinoma is a fast-growing form that grows near
the surface of the lung. An initial diagnosis of large cell
carcinoma is frequently reclassified to squamous cell carcinoma or
adenocarcinoma on further investigation.
[0487] For small cell lung cancer (SCLC), prognosis is also poor.
It tends to start in the larger breathing tubes and grows rapidly
becoming quite large. It is initially more sensitive to
chemotherapy, but ultimately carries a worse prognosis and is often
metastatic at presentation. SCLC is strongly associated with
smoking.
[0488] Other types of lung cancer include carcinoid lung cancer,
adenoid cystic carcinoma, cylindroma, mucoepidermoid carcinoma, and
metastatic cancers which originate in other parts of the body and
metatisize to the lungs. Generally, these cancers are identified by
the site of origin, i.e., a breast cancer metastasis to the lung is
still known as breast cancer. Conversely, the adrenal glands,
liver, brain, and bone are the most common sites of metastasis from
primary lung cancer itself
[0489] Due to the poor prognosis for lung cancer sufferers, early
detection is of paramount importance. However, the screening
methodologies currently widely available have been reported to be
largely ineffective. Regular chest radiography and sputum
examination programs were not effective in reducing mortality from
lung cancer, leading the authors to conclude that the current
evidence did not support screening for lung cancer with chest
radiography or sputum cytology, and that frequent chest x-ray
screening might be harmful. (See Manser R L, et al., Screening for
lung cancer. Cochrane Database of Systematic Reviews 2004, Issue 1.
Art. No.: CD001991. DOI: 10.1002/14651858.CD01991.pub2.).
[0490] Computed tomography (CT) scans can uncover tumors not yet
visible on an X-ray. CT scanning is now being actively evaluated as
a screening tool for lung cancer in high risk patients. In a study
of over 31,000 high-risk patients, 85% of the 484 detected lung
cancers were stage I and were considered highly treatable (see
Henschke C I, et al., Survival of patients with stage I lung cancer
detected on CT screening. N Engl J. Med., 355(17):1763-71,
(2006).
[0491] In contrast, a recent study in which 3,200 current or former
smokers were screened for 4 years and offered 3 or 4 CT scans
reported increased diagnoses of lung cancer and increased
surgeries, but no significant differences between observed and
expected numbers of advanced cancers or deaths (see Bach P B, et
al., Computed Tomography Screening and Lung Cancer Outcomes, JAMA.,
297:953-961 (2007)).
[0492] It should be noted that screening studies have only been
done in high risk populations, such as smokers and workers with
occupational exposure to certain substances. A more definitive
appraisal of the efficacy of screening using CT may need await the
results of ongoing randomized trials in the U.S. and Europe. This
is important when one considers that repeated radiation exposure
from screening could actually induce carcinogenesis in a small
percentage of screened subjects, so this risk should be mitigated
by a (relatively) high prevalence of lung cancer in the population
being screened. This high prevalence can be achieved by
prescreening prior to CT scanning by, for example, the methods
described herein.
[0493] The invention will now be described in more detail, with
reference to the following non-limiting examples.
Example 1
Case Association Study
Introduction
[0494] Case-control association studies allow the careful selection
of a control group where matching for important risk factors is
critical. In this study, smokers diagnosed with lung cancer and
smokers without lung cancer with normal lung function were
compared. This unique control group is highly relevant as it is
impossible to pre-select smokers with zero risk of lung
cancer--i.e., those who although smokers will never develop lung
cancer. Smokers with a high pack year history and normal lung
function were used as a "low risk" group of smokers, as the
Applicants believe it is not possible with current knowledge to
identify a lower risk group of smokers. The Applicants believe,
without wishing to be bound by any theory, that this approach
allows for a more rigorous comparison of low penetrant, high
frequency polymorphisms that may confer an increased risk of
developing lung cancer. The Applicants also believe, again without
wishing to be bound by any theory, that there may be polymorphisms
that confer a degree of protection from lung cancer which may only
be evident if a smoking cohort with normal lung function is
utilised as a comparator group. Thus smokers with lung cancer would
be expected to have a lower frequency of these polymorphisms
compared to smokers with normal lung function and no diagnosed lung
cancer.
Methods
Subject Recruitment
[0495] Subjects of European decent who had smoked a minimum of
fifteen pack years and diagnosed with lung cancer were recruited.
Subjects met the following criteria: diagnosed with lung cancer
based on radiological and histological grounds, including primary
lung cancers with histological types of small cell lung cancer,
squamous cell lung cancer, adenocarinoma of the lung, non-small
cell cancer (where histological markers can not distinguish the
subtype) and broncho-alveolar carcinoma. Subjects could be of any
age and at any stage of treatment after the diagnosis had been
confirmed. 239 subjects were recruited, of these 53% were male, the
mean FEV1/FVC (1 SD) was 61% (14), mean FEV 1 as a percentage of
predicted was 71 (22). Mean age, cigarettes per day and pack year
history was 69 yrs (11), 18 cigarettes/day (11) and 38 pack years
(31), respectively. 484 European subjects who had smoked a minimum
of twenty pack years and who had never suffered breathlessness and
had not been diagnosed with an obstructive lung disease or lung
cancer in the past were also studied. This control group was
recruited through clubs for the elderly and consisted of 60% male,
the mean FEV1/FVC (1 SD) was 76% (8), mean FEV1 as a percentage of
predicted was 101 (10). Mean age, cigarettes per day and pack year
history was 60 yrs (12), 24 cigarettes/day (12) and 41 pack years
(25), respectively. Using a PCR based method (Sandford et al.,
1999), all subjects were genotyped for the .alpha.1-antitrypsin
mutations (S and Z alleles) and those with the ZZ allele were
excluded. On regression analysis, the age difference and pack years
difference observed between lung cancer sufferers and resistant
smokers was found not to determine FEV or lung cancer.
[0496] This study shows that polymorphisms found in greater
frequency in lung cancer patients compared to resistant smokers may
reflect an increased susceptibility to the development of lung
cancer. Similarly, polymorphisms found in greater frequency in
resistant smokers compared to lung cancer may reflect a protective
role.
Summary of Characteristics for the Lung Cancer Subjects and
Resistant Smokers.
TABLE-US-00002 [0497] Parameter: Lung Cancer Resistant smokers Mean
(1 SD) N = 239 N = 484 Differences % male 53% 60% ns Age (yrs) 69
(11) 60 (12) P < 0.05 Pack years 38 (31) 41 (25) P < 0.05
Cigarettes/day 18 (11) 24 (12) ns FEV1 (L) 1.8 (0.6) 2.8 (0.7) P
< 0.05 FEV1 % predict 71 (22) 101% (10) P < 0.05 FEV1/FVC 61
(14) 76 (8) P < 0.05 Means and 1 SD
Polymorphism Genotyping using the Sequenom Autoflex Mass
Spectrometer
[0498] Genomic DNA was extracted from whole blood samples
(Maniatis, T., Fritsch, E. F. and Sambrook, J., Molecular Cloning
Manual. 1989). Purified genomic DNA was aliquoted (10 ng/ul
concentration) into 96 well plates and genotyped on a Sequenom.TM.
system (Sequenom.TM. Autoflex Mass Spectrometer and Samsung 24 pin
nanodispenser) using the following sequences, amplification
conditions and methods.
[0499] The following conditions were used for the PCR multiplex
reaction: final concentrations were for 10.times.Buffer 15 mM
MgCl.sub.2 1.25.times., 25 mM MgCl.sub.2 1.625 mM, dNTP mix 25 mM
500 uM, primers 4 uM 100 nM, Taq polymerase (Quiagen hot start)
0.15 U/reaction, Genomic DNA 10 ng/ul. Cycling times were
95.degree. C. for 15 min, (5.degree. C. for 15 s, 56.degree. C.
30s, 72.degree. C. 30s for 45 cycles with a prolonged extension
time of 3 min to finish. We used shrimp alkaline phosphotase (SAP)
treatment (2 ul to 5 ul per PCR reaction) incubated at 35.degree.
C. for 30 min and extension reaction (add 2 ul to 7 ul after SAP
treatment) with the following volumes per reaction of: water, 0.76
ul; hME 10.times. termination buffer, 0.2 ul; hME primer (10 uM), 1
ul; MassEXTEND enzyme, 0.04 ul.
TABLE-US-00003 TABLE 1 Sequenom conditions for genotyping SNP_ID
2nd-PCRP 1st-PCRP rs11571833 ACGTTGGATGCTGAATTCTCCTCAGATGAC
[SEQ.ID.NO.1] ACGTTGGATGAATGCAAGTTCTTCGTCAGC [SEQ.ID.NO.2]
rs7214723 ACGTTGGATGAAAACTCAGACACCAGGAGC [SEQ.ID.NO.3]
ACGTTGGATGAGATCAAGAATGAGCCCGTG [SEQ.ID.NO.4] rs10115703
ACGTTGGATGCCTCTTATTTCAGCTGCTGG [SEQ.ID.NO.5]
ACGTTGGATGAGAGAACTCTGATTCTGGCG [SEQ.ID.NO.6] rs2306022
ACGTTGGATGACCTTGCCCGTGTGGTTGAA [SEQ.ID.NO.7]
ACGTTGGATGTGGCAGGGTACACAGTCACA [SEQ.ID.NO.8] rs1056503
ACGTTGGATGCTGCTGTTTCTCAGAGTTTC [SEQ.ID.NO.9]
ACGTTGGATGGCCTGATTCTTCACTACCTG [SEQ.ID.NO.10] rs2273953
ACGTTGGATGTGCTCAGGTGTCATTCCTTC [SEQ.ID NO.26]
ACGTTGGATGGGTGGACTGGGCCATCTTC [SEQ.ID.NO.27] c74delA
ACGTTGGATGTTCTGTAACCTGGCTTTCTC [SEQ.ID.NO.28]
ACGTTGGATGCCAGGAATTCCCAGCTTCTT [SEQ.ID.NO.29] rs1799732
ACGTTGGATGCAAAACAAGGGATGGCGGAA [SEQ.ID.NO.30]
ACGTTGGATGAAAGGAGCTGTACCTCCTCG [SEQ.ID.NO.31] rs2279115
ACGTTGGATGATCAGAAGAGGATTCCTGCC [SEQ.ID.NO.32]
ACGTTGGATGTTCACGCCTCCCCAGGAGA [SEQ.ID.NO.33] rs2317676
ACGTTGGATGTATGAACTGGGAGATGCTGG [SEQ.ID.NO.34]
ACGTTGGATGTGTTGGGAGTGAGGATGTCT [SEQ.ID.NO.35] rs5743836
ACGTTGGATGTTGGGATGTGCTGTTCCCTC [SEQ.ID.NO.36]
ACGTTGGATGAGCAGAGACATAATGGAGGC [SEQ.ID.NO.37] rs6413429
ACGTTGGATGTGTCAGGAGGCCTTCAGGTG [SEQ.ID.NO.38]
ACGTTGGATGGTTTTATGAGGGCACTGGTC [SEQ.ID.NO.39] rs1139417
ACGTTGGATGAGGCCATAGCTGTCTGGCAT [SEQ.ID NO.40]
ACGTTGGATGTTCCCTTTGTCCCTGGTCT [SEQ.ID.NO.41] rs763110
ACGTTGGATGAGGCTGCAAACCAGTGGAAC [SEQ.ID.NO.42]
ACGTTGGATGCTGGGCAAACAATGAAAATG [SEQ.ID.NO.43] SNP_ID AMP_LEN
UP_CONF MP_CONF Tm(NN) PcGC PWARN UEP_DIR UEP_MASS rs11571833 109
96.8 69.1 46.3 44.4 F 5409.5 rs7214723 113 99.3 69.1 61.3 58.3 dH F
7304.7 rs10115703 101 98.7 69.1 59 50 R 7884.1 rs2306022 111 91.8
90.9 53.8 68.8 D R 4867.2 rs1056503 104 98.5 90.9 48 42.1 R 5775.8
rs2273953 98 90.6 90.6 49.3 58.8 H R 5137.3 c74delA 101 94.9 69.7
45.7 25 D F 7295.8 rs1799732 99 97.3 66.7 59.5 66.7 d F 6183
rs2279115 99 88.3 78.5 52.3 64.7 d F 5073.3 rs2317676 97 98.7 66.7
63.3 62.5 DH R 7298.7 rs5743836 100 98.6 88.1 53 64.7 R 5104.3
rs6413429 93 94.2 66.7 56.5 70.6 D F 5196.4 rs1139417 99 92.2 99.6
56.2 70.6 d F 5098.3 rs763110 92 92.8 66.7 56.3 44 d R 7591.9
EXT1.sub.-- EXT1.sub.-- SNP_ID UEP_SEQ CALL MASS EXT1_SEQ
rs11571833 CCTCAGATGACTCCATTT [SEQ.ID.NO.11] A 5680.7
CCTCAGATGACTCCATTTA [SEQ.ID.NO.12] rs7214723
TGTTCCCCTGGGTGGACAACTCAC [SEQ.ID.NO.13] C 7551.9
TGTTCCCCTGGGTGGACAACTC [SEQ.ID.NO.14] ACC rs10115703
TACTCCTGCCTCTAGGAAAGACCACA [SEQ.ID.NO.15] G 8131.3
TACTCCTGCCTCTAGGAAAGAC [SEQ.ID.NO.16] CACAC rs2306022
CCCTGCCTGGAGGACA [SEQ.ID.NO.17] G 5114.4 CCCTGCCTGGAGGACAC
[SEQ.ID.NO.18] rs1056503 CTGAGATGTGCTCCTTTTT [SEQ.ID.NO.19] G
6022.9 CTGAGATGTGCTCCTTTTTC [SEQ.ID.NO.20] rs2273953
CTTCCTTCCTGCAGAGG [SEQ.ID.NO.44] T 5408.6 CTTCCTTCCTGCAGAGGA
[SEQ.ID.NO.45] c74delA GGCTTTCTCTTTTATTTTATAGTT [SEQ.ID.NO.46] C
7542.9 GGCTTTCTCTTTTATTTTATAG [SEQ.ID.NO.47] TTC rs1799732
CCCAACCCCTCCTACCCGTTC [SEQ.ID.NO.48] C 6430.2
CCCAACCCCTCCTACCCGTTCC [SEQ.ID.NO.49] rs2279115 GGCTCCTTCATCGTCCC
[SEQ.ID.NO.50] C 5320.5 GGCTCCTTCATCGTCCCC [SEQ.ID.NO.51] rs2317676
GATGCTGGTACATCCCCCAGGCCA [SEQ.ID.NO.52] G 7545.9
GATGCTGGTACATCCCCCAGGC [SEQ.ID.NO.53] CAC rs5743836
GCTGTTCCCTCTGCCTG [SEQ.ID.NO.54] T 5375.5 GCTGTTCCCTCTGCCTGA
[SEQ.ID NO.55] rs641 3429 GGAGGGCTCCACCCTGA [SEQ.ID.NO.56] G 5483.6
GGAGGGCTCCACCCTGAG [SEQ.ID.NO.57] rs1139417 CCTGACCTGCTGCTGCC
[SEQ.ID.NO.58] A 5369.5 CCTGACCTGCTGCTGCCA [SEQ.ID.NO.59] rs763110
AACCCACAGAGCTGCTTTGTATTTC [SEQ.ID.NO.60] T 7863.2
AACCCACAGAGCTGCTTTGTAT [SEQ.ID.NO.61] TTCA EXT2 EXT2 SNP_ID CALL
MASS EXT2_SEQ rs11571833 T 5736.6 CCTCAGATGACTCCATTTT
[SEQ.ID.NO.21] rs7214723 T 7631.8 TGTTCCCCTGGGTGGACAACTCACT
[SEQ.ID.NO.22] rs10115703 A 8211.2 TACTCCTGCCTCTAGGAAAGACCACAT
[SEQ.ID.NO.23] rs2306022 A 5194.3 CCCTGCCTGGAGGACAT [SEQ.ID.NO.24]
rs1056503 T 6047 CTGAGATGTGCTCCTTTTTA [SEQ.ID.NO.25] rs2273953 C
5424.6 CTTCCTTCCTGCAGAGGG [SEQ.ID.NO.62] c74delA A 7567
GGCTTTCTCTTTTATTTTATAGTTA [SEQ. ID. NO.63] rs1799732 DEL 6454.2
CCCAACCCCTCCTACCCGTTCA [SEQ.ID.NO.64] rs2279115 A 5344.5
GGCTCCTTCATCGTCCCA [SEQ.ID.NO.65] rs2317676 A 7625.8
GATGCTGGTACATCCCCCAGGCCAT [SEQ. ID.NO.66] rs5743836 C 5391.5
GCTGTTCCCTCTGCCTGG [SEQ.ID.NO.67] rs6413429 T 5523.5
GGAGGGCTCCACCCTGAT [SEQ.ID.NO.68] rs1139417 G 5385.5
CCTGACCTGCTGCTGCCG [SEQ.ID.NO.69] rs763110 C 7879.2
AACCCACAGAGCTGCTTTGTATTTCG [SEQ.ID.NO.70] EXT3 EXT3 SNP ID CALL
MASS EXT3_SEQ c74delA G 7583 GGCTTTCTCTTTTATTTTATAGTTG
[SEQ.ID.NO.71] EXT4 EXT4 SNP_ID CALL MASS EXT4_SEQ c74delA T 7622.8
GGCTTTCTCTTTTATTTTATAGTTT [SEQ.ID.NO.72]
Results
Univariate Analyses:
TABLE-US-00004 [0500] TABLE 2 Cerberus 1 (Cer 1) R19W A/G (rs
10115703) polymorphism allele and genotype frequencies in the Lung
cancer patients and resistant smokers. Allele* Genotype Frequency A
G AA AG GG Lung Cancer 47 (10%) 421 (90%) 2 (1%) 43 (18%) 189 (81%)
n = 234 (%) Resistant 66 (7%) 878 (93%) 7 (1%) 52 (11%) 413 (88%) n
= 472 (%) *number of chromosomes (2n)
Genotype. AA/AG vs GG for lung cancer vs resistant, Odds ratio
(OR)=1.7, 95% confidence limits 1.1-2.6, .chi..sup.2 (Yates
uncorrected)=5.63, p=0.02,
[0501] AA/AG genotype=susceptibility (GG protective) [0502] Allele.
A vs G for lung cancer vs resistant, Odds ratio (OR)=1.5, 95%
confidence limits 1.0-2.2, .chi..sup.2 (Yates uncorrected)=3.95,
p=0.05,
[0503] A allele=susceptibility
TABLE-US-00005 TABLE 3 XRCC4 Ser307Ser G/T (rs1056503) polymorphism
allele and genotype frequencies in the Lung cancer patients and
resistant smokers. Allele* Genotype Frequency G T GG GT TT Lung
Cancer 68 (15%) 374 (85%) 8 (4%) 52 (24%) 161 (72%) n = 221 (%)
Resistant 66 (11%) 838 (89%) 5 (1%) 98 (21%) 370 (78%) n = 473 (%)
*number of chromosomes (2n)
Genotype. GG/GT vs TT for lung cancer vs resistant, Odds ratio
(OR)=1.3, 95% confidence limits 0.9-2.0, .chi..sup.2 (Yates
uncorrected)=2.4, p=0.12,
[0504] GG/GT genotype=susceptibility (TT protective)
Allele. G vs T for lung cancer vs resistant, Odds ratio (OR)=1.4,
95% confidence limits 1.0-2.0, .chi..sup.2 (Yates
uncorrected)=4.28, p=0.04,
[0505] G allele=susceptibility
TABLE-US-00006 TABLE 4 BRCA2 K3326X A/T (rs 11571833) polymorphism
allele and genotype frequencies in the Lung cancer patients and
resistant smokers. Allele* Genotype Frequency A T AA AT TT Lung
Cancer 450 (97%) 12 (3%) 220 (95%) 10 (4%) 1 (0.4%) n = 231 (%)
Resistant 915 (99%) 9 (1%) 453 (98%) 9 (2%) 0 (0%) n = 462 (%)
*number of chromosomes (2n)
Genotype. AT/TT vs AA for lung cancer vs resistant, Odds ratio
(OR)=2.5, 95% confidence limits 1.0-6.7, .chi..sup.2 (Yates
uncorrected)=4.34, p=0.04, AT/TT genotype=susceptibility (AA
protective) Allele. T vs A for lung cancer vs resistant, Odds ratio
(OR)=2.7, 95% confidence limits 1.1-7.0, .chi..sup.2 (Yates
uncorrected)=5.44, p=0.02, T allele=susceptibility
TABLE-US-00007 TABLE 5 Integrin alpha-11 V433M A/G (rs 2306022)
polymorphism allele and genotype frequencies in the Lung cancer
patients and resistant smokers. Allele* Genotype Frequency A G AA
AG GG Lung Cancer 60 (13%) 406 (87%) 12 (5%) 36 (15%) 185 (79%) n =
233 (%) Resistant 89 (9%) 863 (91%) 6 (1%) 77 (16%) 393 (83%) n =
476 (%) *number of chromosomes (2n)
Genotype. AA vs AG/GG for lung cancer vs resistant, Odds ratio
(OR)=4.3, 95% confidence limits 1.5-12.9, .chi..sup.2 (Yates
uncorrected)=9.55, p=0.002,
[0506] AA genotype=susceptibility
Allele. A vs G for lung cancer vs resistant, Odds ratio (OR)=1.4,
95% confidence limits 1.0-2.1, X.sup.2 (Yates uncorrected)=4.14,
p=0.04,
[0507] A allele=susceptibility
TABLE-US-00008 TABLE 6 CAMKK1 Calcium/calmodulin-dependent protein
kinase kinase 1 E375G T/C (rs7214723) polymorphism allele and
genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency T C TT TC CC Lung Cancer 239
(51%) 227 (49%) 62 (26%) 115 (49%) 56 (24%) n = 233 (%) Resistant
514 (56%) 412 (44%) 149 (32%) 216 (47%) 98 (21%) n = 463 (%)
*number of chromosomes (2n)
Genotype. TT vs TC/CC for lung cancer vs resistant, Odds ratio
(OR)=0.76, 95% confidence limits 0.5-1.1, .chi..sup.2 (Yates
uncorrected)=2.27, p=0.13,
[0508] TT genotype=protective
Allele. T vs C for lung cancer vs resistant, Odds ratio (OR)=0.84,
95% confidence limits 0.7-1.1, .chi..sup.2 (Yates
uncorrected)=2.22, p=0.14,
[0509] T allele=protective
TABLE-US-00009 TABLE 7 P73 C/T (rs 2273953) polymorphism allele and
genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency C T CC CT TT Lung Cancer 316
(69%) 142 (31%) 99 (43%) 118 (52%) 12 (5%) n = 229 (%) Resistant
742 (78%) 206 (22%) 295 (62%) 152 (32%) 27 (6%) n = 474 (%) *number
of chromosomes (2n)
Genotype. CC vs CT/TT for lung cancer vs resistant, Odds ratio
(OR)=0.46, 95% confidence limits 0.33-0.64, .chi..sup.2 (Yates
uncorrected)=22.0, p<0.001,
[0510] CC genotype=protective (CT/TT susceptible)
Allele. C vs T for lung cancer vs resistant, Odds ratio (OR)=0.62,
95% confidence limits0.48-0.80, .chi..sup.2 (Yates corrected)=14.0,
p<0.001,
[0511] C allele=protective
TABLE-US-00010 TABLE 8 CYP 3A43 A/T c74delA polymorphism allele and
genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency A T AA AT TT Lung Cancer 442
(94%) 26 (6%) 209 (89%) 24 (10%) 1 (0.5%) n = 234 (%) Resistant 935
(97%) 31 (3%) 452 (94%) 31 (6%) 0 (0%) n = 483 (%) *number of
chromosomes (2n)
Genotype. AT/TT vs AA for lung cancer vs resistant, Odds ratio
(OR)=1.74, 95% confidence limits 0.97-3.13, .chi..sup.2=(Yates
uncorrected)=4.0, p=0.05,
[0512] AT/TT genotype susceptible
Allele. T vs A for lung cancer vs resistant, Odds ratio (OR)=1.8,
95% confidence limits 1-3.1, X.sup.2 (Yates uncorrected)=4.54,
p=0.03,
[0513] T allele=susceptible
TABLE-US-00011 TABLE 9 BCL2 A/C (rs 2279115) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency A C AA AC CC Lung Cancer 223
(47%) 249 (53%) 55 (23%) 113 (48%) 68 (29%) n = 236 (%) Resistant
513 (54%) 445 (46%) 146 (31%) 221 (46%) 112 (23%) n = 479 (%)
*number of chromosomes (2n)
Genotype. AA vs AC/CC for lung cancer vs resistant, Odds ratio
(OR)=0.69, 95% confidence limits 0.48-1.0, .chi..sup.2 (Yates
uncorrected)=4.0, p=0.05,
[0514] AA genotype=protective
Allele. A vs C for lung cancer vs resistant, Odds ratio (OR)=0.78,
95% confidence limits 0.62-0.97, .chi..sup.2 (Yates corrected)=5.0,
p=0.02,
[0515] A allele=protective
TABLE-US-00012 TABLE 10 ITGB3 A/G (rs 2317676) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency A G AA AG GG Lung Cancer 445
(95%) 23 (5%) 211 (90%) 23 (10%) 0 (0%) n = 234 (%) Resistant n =
484 884 (91%) 84 (9%) 406 (84%) 72 (15%) 6 (1%) (%) *number of
chromosomes (2n)
Genotype. AG/GG vs AA for lung cancer vs resistant, Odds ratio
(OR)=0.57, 95% confidence limits 0.34-0.95, .chi..sup.2 (Yates
uncorrected)=5.2, p=0.02,
[0516] AG/GG genotype=protective
Allele. G vs A for lung cancer vs resistant, Odds ratio (OR)=0.54,
95% confidence limits0.33-0.89, X.sup.2 (Yates uncorrected)=6.5,
p=0.01, G allele=protective Integrin beta 3 is also referred to as
platelet glycoprotein IIIa or antigen CD61.
TABLE-US-00013 TABLE 11 DAT1 G/T (rs 6413429) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency G T GG GT TT Lung Cancer 427
(92%) 37 (8%) 195 (84%) 37 (16%) 0 (0%) n = 232 (%) Resistant n =
485 914 (94%) 56 (6%) 433 (89%) 48 (10%) 4 (1%) (%) *number of
chromosomes (2n)
Genotype. TT/GT vs GG for lung cancer vs resistant, Odds ratio
(OR)=1.6, 95% confidence limits 1.0-2.6, .chi..sup.2 (Yates
uncorrected)=3.9, p=0.05,
[0517] TT/GT genotype=susceptible
Dopamine transporter 1 (DAT1) is also known as solute carrier
family 6 (neurotransmitter transporter, dopamine), member 3
(SLC6A3).
TABLE-US-00014 TABLE 12 TNFR1 A/G (rs1139417) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency A G AA AG GG Lung Cancer 277
(62%) 171 (38%) 87 (39%) 103 (46%) 34 (15%) n = 224 (%) Resistant
536 (56%) 420 (44%) 143 (30%) 250 (52%) 85 (18%) n = 478 (%)
*number of chromosomes (2n)
Genotype. AA vs AG/GG for lung cancer vs resistant, Odds ratio
(OR)=1.5, 95% confidence limits 1-2.1, .chi..sup.2 (Yates
uncorrected)=5.5, p=0.02,
[0518] AA genotype=susceptible
Allele. A vs G for lung cancer vs resistant, Odds ratio (OR)=1.3,
95% confidence limits 1.0-1.6, .chi..sup.2 (Yates uncorrected)=4.2,
p=0.04,
[0519] A allele=susceptible
TABLE-US-00015 TABLE 13 DRD2 C/Del (rs 1799732) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency C Del CC CDel DelDel Lung
Cancer 426 (92%) 36 (8%) 197 (85%) 32 (14%) 2 (1%) n = 231 (%)
Resistant 857 (89%) 109 (11%) 376 (78%) 105 (22%) 2 (0.5%) n = 483
(%) *number of chromosomes (2n)
Genotype. CDel/DelDel vs CC for lung cancer vs resistant, Odds
ratio (OR)=0.61, 95% confidence limits 0.39-0.94, .chi..sup.2
(Yates uncorrected)=5.4, p=0.02,
[0520] CDel/DelDel genotype=protective
Allele. Del vs C for lung cancer vs resistant, Odds ratio
(OR)=0.66, 95% confidence limits 0.44-1.0, .chi..sup.2 (Yates
uncorrected)=4.2, p=0.04,
[0521] Del=protective
TABLE-US-00016 TABLE 14 FasL C/T (rs 763110) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency C T CC CT TT Lung Cancer 302
(66%) 156 (34%) 97 (42%) 108 (47%) 24 (11%) n = 229 (%) Resistant
596 (61%) 374 (39%) 189 (39%) 218 (45%) 78 (16%) n = 485 (%)
*number of chromosomes (2n)
Genotype. TT vs CC/CT for lung cancer vs resistant, Odds ratio
(OR)=0.61, 95% confidence limits 0.36-1.0, .chi..sup.2 (Yates
uncorrected)=4.0, p=0.05,
[0522] TT genotype=protective
Fas ligand (TNF superfamily, member 6) is also known as FASLG, CD
178, CD95L, TNFSF6, and APT1LG1.
TABLE-US-00017 TABLE 15 TLR9 C/T (rs 5743836) polymorphism allele
and genotype frequencies in the Lung cancer patients and resistant
smokers. Allele* Genotype Frequency T C TT TC CC Lung Cancer 386
(84%) 76 (16%) 164 (71%) 58 (25%) 9 (4%) n = 231 (%) Resistant 791
(85%) 139 (15%) 332 (71%) 127 (27%) 6 (1%) n = 465 (%) *number of
chromosomes (2n)
Genotype. CC vs TC/TT for lung cancer vs resistant, Odds ratio
(OR)=3.1, 95% confidence limits 1.0-9.9, .chi..sup.2 (Yates
uncorrected)=5.0, p=0.03,
[0523] CC genotype=susceptible
TABLE-US-00018 TABLE 16 Summary table of protective and
susceptibility polymorphisms for lung cancer. Gene and SNP rs
number Genotype Phenotype OR P value Cerberus 1 (Cer 1) R19W
A/G.sup.1 rs10115703 AA/AG susceptiblility 1.7 0.02 XRCC4 Ser307Ser
G/T.sup.1 rs1056503 GG/GT susceptiblility 1.3 0.04 BRCA2 K3326X
A/T.sup.1 rs11571833 AT/TT susceptiblility 2.5 0.04 Integrin
alpha-11 V433M A/G.sup.1 rs2306022 AA susceptiblility 4.3 0.002
CAMKK1 E375G T/C.sup.1 rs7214723 TT protective 0.76 0.13 P73
rs2273953 CC protective 0.46 <0.001 CYP3A43 C74 delA AT/TT
susceptiblility 1.74 0.05 BCL2 rs2279115 AA protective 0.69 0.05
ITGB3 rs2317676 AG/GG protective 0.57 0.02 DAT1 rs6413429 GT/TT
susceptibility 1.6 0.05 TNFR1 rs1139417 AA susceptibility 1.5 0.02
DRD2 rs1799732 CDel/DelDel protective 0.61 0.02 FasL rs763110 TT
protective 0.61 0.05 TLR9 rs5743836 CC susceptibility 3.1 0.03
.sup.1included in the 5 SNP panel described below. Odds ratios and
P values derived from univariate analyses described above.
[0524] SNP scores for each subject were derived by assigning a
score of +1 for the presence of susceptiblility genotypes or -1 for
the presence of protective genotypes of the 5 SNPs included in the
panel as identified in Table 16 above. The scores are added to
derive the total SNP score for each subject. Table 17 below shows
the distribution of SNP scores derived from the 5 SNP panel amongst
the lung cancer patients and the resistant smoker controls.
TABLE-US-00019 TABLE 17 Distribution of SNP scores (5 SNP panel) in
smoker with and without lung cancer. Lung cancer SNP score - 5 SNP
panel Cohort -1 0 1 2 Lung cancer N = 239 (%) 33 (14%) 119 (50%) 75
(31%) 12 (5%) Control smokers N = 484 (%) 104 (21%) 264 (54%) 100
(21%) 16 (3%) % with lung cancer 33/137 (24%) 119/383 (31%) 75/175
(43%) 12/28 (43%)
[0525] The likelihood of having lung cancer according to the lung
cancer SNP score generated from the 5 SNP panel is shown
graphically in FIG. 1. The log odds of having lung cancer according
to the SNP score derived from the 5 SNP panel presented in Table 17
is shown in FIG. 2.
Example 2
[0526] This example presents an analysis of distributions of SNP
scores derived for lung cancer sufferers and control resistant
smokers using the polymorphisms described in Table 18 below. Table
18 presents a summary of selected protective and susceptibility
SNPs identified in PCT/NZ2006/000125 (published as WO2006/123955)
and related applications (New Zealand Patent Application No.s
540203/541787/543297), and herein that were included in additional
panels of SNPs.
[0527] SNPs 1-11 identified in Table 18 were included in both the
11 SNP panel A and the 16 SNP panel used to generate SNP scores as
discussed below. SNPs 12-16 identified in Table 18 were included in
both the 5 SNP panel described in Example 1 above, and in the 16
SNP panel used to generate SNP scores as discussed below. Odd's
ratios (OR) and p values are for cancer patients compared to
resistant smokers with normal lung function.
TABLE-US-00020 TABLE 18 Summary of selected protective and
susceptibility polymorphisms P SNP# Gene Polymorphism Genotype
Phenotype OR value 1 Interleukin-18 (IL-18) -133 G/C CG/GG
protective 1.5 0.09 CC susceptibility 2 CYP2E1 -1053 C/T (Rsa I)
TT/TC susceptibility 1.9 0.13 3 N-acetyltransferase 2 Arg 197 Gln
A/G GG susceptibility 1.5 0.08 (NAT2) 4 Interleukin 1B (IL-1B) -511
A/G GG susceptibility 1.6 0.04 5 Anti-chymotrypsin Ala 15 Thr GG
susceptibility 1.7 0.06 (ACT) 6 .alpha.1-antitrypsin S allele.sup.1
AT/TT susceptibility 7 Interleukin-8 (IL-8) -251 A/T AA protective
4.1 0.002 8 XPD Lys-751 Gln G/T GG protective 1.7 0.18 9 Superoxide
dismutase 3 Arg 312 Gln (+760 CG/GG protective 3.38 0.03 (SOD3)
G/C) 10 REV1 Phe 257 Ser C/T CC protective 0.73 0.20 11
.alpha.1-antitrypsin Z allele.sup.1 AG protective 12 Cerberus 1
(Cer 1) R19W A/G.sup.2 AA/AG susceptiblility 1.7 0.02 (rs 10115703)
13 XRCC4 Ser307Ser G/T.sup.2 GG/GT susceptiblility 1.3 0.04
(rs1056503) 14 BRCA2 K3326X A/T.sup.2 AT/TT susceptiblility 2.5
0.04 (rs 11571833) 15 Integrin alpha-11 V433M A/G.sup.2 AA
susceptiblility 4.3 0.002 (rs 2306022) 16 CAMKK1 E375G T/C.sup.2 TT
protective 0.76 0.13 (rs7214723) .sup.1discussed in PCT
International application PCT/NZ2006/000125. .sup.2included in both
the 5 SNP panel (described in Example 1) and the 16 SNP panel.
[0528] Table 19 below presents the distribution of SNP scores
derived from the 11 SNP panel A consisting of SNPs numbers 1 to 11
from Table 18 in the lung cancer patients and the resistant smoker
controls.
TABLE-US-00021 TABLE 19 Distribution of the lung cancer SNP score
##STR00001##
[0529] The shaded SNP scores (0, 1, and 2) can be viewed as low to
average risk of lung cancer. At this threshold (cut-off), 7% of
lung cancer cases were present, while 29% of the control smokers
were present. On the graph plotting lung cancer frequency versus
SNP score (FIG. 3), this equates to an approximately 10% risk of
lung cancer. This is the average across all smokers. The likelihood
of having lung cancer according to the SNP score derived from the
11 SNP panel A is shown in FIG. 3.
[0530] The distribution of SNP scores among lung cancer patients
and resistant smoker controls were further analysed as follows.
FIG. 4 depicts a receiver-operator curve analysis with sensitivity
and sensitivity for the lung cancer 11 SNP panel A. This was
developed according to the model:
TABLE-US-00022
(IL18_133_S+CYP2E1_Rsa1_S+NAT2_197_S+IL1B_511_S+ACT_15_S+s_allele_S+
IL8_251_S+z_allele_s) - (XPD_751_P+SOD3_213_P+REV1_257_P) if age
> 60 then add 4 if FHx lung Ca then add 3 Area under the ROC
curve Results Area 0.7483 Std. Error 0.01907 95% confidence
interval 0.7109 to 0.7856 P value <0.0001 Cutoff Sensitivity 95%
Cl Specificity 95% Cl Likelihood ratio >-0.5000 0.9958 0.9769 to
0.9999 0.004132 0.0005008 to 0.01485 1.00 >0.5000 0.9916 0.9701
to 0.9990 0.04752 0.03036 to 0.07045 1.04 >1.500 0.9707 0.9406
to 0.9881 0.1405 0.1108 to 0.1747 1.13 >2.500 0.9331 0.8936 to
0.9613 0.2934 0.2532 to 0.3362 1.32 >3.500 0.8828 0.8351 to
0.9207 0.4360 0.3913 to 0.4814 1.57 >4.500 0.8285 0.7746 to
0.8740 0.5351 0.4896 to 0.5803 1.78 >5.500 0.7406 0.6801 to
0.7950 0.6405 0.5960 to 0.6833 2.06 >6.500 0.5439 0.4785 to
0.6083 0.7810 0.7415 to 0.8171 2.48 >7.500 0.3598 0.2990 to
0.4242 0.9008 0.8707 to 0.9260 3.63 >8.500 0.2050 0.1557 to
0.2618 0.9649 0.9444 to 0.9794 5.84 >9.500 0.1046 0.06884 to
0.1505 0.9938 0.9820 to 0.9987 16.88 >10.50 0.03766 0.01736 to
0.07028 0.9979 0.9885 to 0.9999 18.23 >11.50 0.004184 0.0001059
to 0.02309 1.000 0.9924 to 1.000
[0531] FIG. 5 herein presents a graph showing the distribution of
SNP score derived from the 11 SNP panel A among lung cancer
sufferers and among resistant smoker controls.
TABLE-US-00023 TABLE 20 Distribution of the lung cancer SNP score
derived from the 16 SNP panel ##STR00002##
[0532] The shaded SNP scores (<1, 2, and 3) can be viewed as low
to average risk of lung cancer. At this cut-off, 8% of lung cancer
cases were present, while 41% of control smokers were present. On
the graph plotting lung cancer frequency and SNP score (FIG. 6),
this equates to about a 10% risk of lung cancer, the average across
all smokers. The likelihood of having lung cancer according to the
SNP score derived from the 16 SNP panel is shown in FIG. 6.
[0533] The distribution of SNP scores among lung cancer patients
and resistant smoker controls were further analysed as follows.
FIG. 7 depicts a receiver-operator curve analysis with sensitivity
and sensitivity for the lung cancer 16 SNP panel. This was
developed according to the model:
TABLE-US-00024
(IL18_133_S+CYP2E1_Rsa1_S+NAT2_197_S+IL1B_511_S+ACT_15_S+s_allele_S+
IL8_251_S+z_allele_s) -(XPD_751_P+SOD3_213_P+REV1_257_P +
(ITGA11_s+Cer1_s+BRAC2_s +XRCC4_307_s) -CAMKK1_p if age > 60
then add 4 if FHx lung Ca then add 3 Area under the ROC curve
Results Area 0.7621 Std. Error 0.01855 95% confidence interval
0.7257 to 0.7985 P value <0.0001 Cutoff Sensitivity 95% Cl
Specificity 95% Cl Likelihood ratio >-0.5000 0.9958 0.9769 to
0.9999 0.01240 0.004563 to 0.02679 1.01 >0.5000 0.9874 0.9638 to
0.9974 0.05992 0.04049 to 0.08492 1.05 >1.500 0.9749 0.9462 to
0.9907 0.1529 0.1220 to 0.1881 1.15 >2.500 0.9456 0.9088 to
0.9707 0.2789 0.2394 to 0.3212 1.31 >3.500 0.9121 0.8688 to
0.9448 0.4132 0.3690 to 0.4585 1.55 >4.500 0.8494 0.7976 to
0.8922 0.5310 0.4854 to 0.5762 1.81 >5.500 0.7406 0.6801 to
0.7950 0.6405 0.5960 to 0.6833 2.06 >6.500 0.5858 0.5205 to
0.6489 0.7851 0.7458 to 0.8209 2.73 >7.500 0.4310 0.3673 to
0.4964 0.8781 0.8456 to 0.9059 3.54 >8.500 0.2469 0.1935 to
0.3066 0.9504 0.9271 to 0.9680 4.98 >9.500 0.1255 0.08632 to
0.1743 0.9814 0.9650 to 0.9915 6.75 >10.50 0.05858 0.03239 to
0.09633 0.9938 0.9820 to 0.9987 9.45 >11.50 0.02092 0.006827 to
0.04814 1.000 0.9924 to 1.000
[0534] FIG. 8 herein presents a graph showing the distribution of
SNP score derived from the 16 SNP panel among lung cancer sufferers
and among resistant smoker controls.
Example 3
[0535] This example presents a multivariate analysis using a 9 SNP
panel comprising the polymorphisms described in Table 21 below.
Table 21 summarises the univariate analysis showing protective and
susceptibility SNPs associated with lung cancer as set out in
Tables 7-15. Odd's ratios (OR) and p values are for cancer patients
compared to resistant smokers with normal lung function.
TABLE-US-00025 TABLE 21 Summary of selected polymorphisms - 9 SNP
panel Gene and SNP rs number Genotype Phenotype OR P value P73
rs2273953 CC protective 0.46 <0.001 CYP3A43 AT/TT
susceptiblility 1.74 0.05 C74 delA BCL2 rs2279115 AA protective
0.69 0.05 ITGB3 rs2317676 AG/GG protective 0.57 0.02 DAT1 rs6413429
GT/TT susceptibility 1.6 0.05 TNFR1 rs1139417 AA susceptibility 1.5
0.02 DRD2 rs1799732 CDel/DelDel protective 0.61 0.02 FasL rs763110
TT protective 0.61 0.05 TLR9 rs5743836 CC susceptibility 3.1
0.03
[0536] As described above in respect of the 5, 11, and 16 SNP
panels, a SNP score was determined for each subject from the
univariate data for this 9 SNP panel. The presence of the
susceptibility SNP genotype was scored +1, and the presence of the
protective SNP genotype was scored -1.
[0537] As shown in FIG. 9, a linear relationship was observed when
the SNP score for lung cancer patients and healthy smoking controls
were analysed together and plotted according to the odds of having
lung cancer, where those with the highest scores have the greatest
risk. In this analysis (floating absolute odds ratio), the lowest
SNP score group is referenced as 1. Those with the highest score (5
or more) have an Odds of 13-they are at 13 fold greater likelihood
(or risk) of being diagnosed with lung cancer.
[0538] For each subject, a composite score that defines a
likelihood of being diagnosed with lung cancer was derived. The SNP
score from the 9 SNP panel was combined with scores according to
age (+4 for age over 60 yo) and family history (+3 for having a
first degree relative with lung cancer) for each subject. This
algorithm generated a composite score for each smoker based on
genotype, age and family history of lung cancer. Table 22 below
shows the results of this multivariate analysis using these 9 SNPs,
age and family history.
TABLE-US-00026 TABLE 22 Multivariate analysis ##STR00003##
[0539] FIG. 10 shows the receiver-operator curve analysis for this
composite lung cancer SNP score. The receiver operator curve
analysis shows the area under the ROC curve is 0.73 for these 9
SNPs. This indicates an acceptable level of discrimination.
[0540] When the frequency distribution for the 9 SNP panel SNP
score is compared between lung cancer cases and controls (FIG. 11),
separation of the lung cancer SNP score between cases and controls
is observed. This reflects the ability of the SNP score to
discriminate between high and low risk smokers. This data shows
that SNPs on their own derive modest levels of risk (small Odds
ratios). These SNPs can be analysed in combination to derive a risk
score with clinical utility in discriminating smokers at high and
low risk of lung cancer based on their genotype, and such analyses
can include non-genetic factors such as age and family history.
Example 4
[0541] This example presents a multivariate analysis using an 11
SNP panel (11 SNP panel B) comprising the polymorphisms described
in Table 23 below. Table 23 summarises the univariate analysis
showing protective and susceptibility SNPs associated with lung
cancer as set out herein. Odd's ratios (OR) and p values are for
cancer patients compared to resistant smokers with normal lung
function. Stepwise regression analysis was also performed, and chi
squared values are presented for each polymorphism.
TABLE-US-00027 TABLE 23 Summary of Selected Polymorphisms - 11 SNP
Panel B Lung Smoking Call Univariate Stepwise SNP (rs#) Genotype
cancer controls rate OR P value regression .chi..sup.2 P value
Phenotype Interleukin-18 CC 237 (54%) 208 (45%) 96% 1.4 0.009 10.4
0.001 susceptibility (-133 G/C) CG/GG 201 (46%) 250 (55%) (1.1-1.9)
Interleukin-8 TT 129 (31%) 109 (23%) 96% 1.5 0.005 6.5 0.01
susceptibility (-251 A/T) AT/AA 284 (69%) 367 (77%) (1.1-2.1)
ITGA11 AA 14 (3%) 6 (1%) 98% 2.6 0.04 susceptibility (rs2306022)
GA/GG 422 (97%) 470 (99%) (0.9-7.6) N-acetylcysteine GG 239 (56%)
222 (47%) 97% 1.4 0.006 5.8 0.02 susceptibility transferase 2 AA/AG
189 (44%) 253 (53%) (1.1-1.9) (rs 1799930)
.alpha.1-antichymotrypsin GG 123 (28%) 96 (20%) 98% 1.6 0.004 7.1
0.008 susceptibility (-15 A/G) AG/AA 312 (72%) 383 (80%) (1.2-2.2)
DAT1 GT/TT 64 (15%) 50 (10%) 98% 1.5 0.04 4.2 0.04 susceptibility
(rs6413429) GG 367 (85%) 431 (90%) (1.0-2.3) P73 CC 219 (52%) 292
(62%) 96% 0.65 0.001 11.8 0.0006 protective (rs 2273953) TC/TT 206
(48%) 178 (38%) (0.49-0.85) SOD3 GG/GC 4 (1%) 15 (3%) 96% 0.28 0.02
7.7 0.005 protective (rs1799895) CC 425 (99%) 451 (97%) (0.10-0.90)
ITGB3 GG/GA 44 (10%) 77 (16%) 98% 0.59 0.008 6.6 0.01 protective
(rs2317676) AA 391 (90%) 403 (84%) (0.39-0.89) DRD2 CDel/Del.Del 70
(16%) 107 (22%) 98% 0.68 0.02 7.3 0.007 protective (rs 1799732) CC
359 (84%) 372 (78%) (0.48-0.96) BCL2 AA 103 (24%) 145 (31%) 97%
0.71 0.03 4.2 0.04 protective (rs 2279115) AC/CC 328 (76%) 330 69%)
(0.53-0.97)
[0542] As described above, a SNP score was determined for each
subject from the univeriate data for the 11 SNP panel B. The
presence of the susceptibility SNP genotype was scored +1, and the
presence of the protective SNP genotype was scored -1.
[0543] For each subject, a score that defines a likelihood of being
diagnosed with lung cancer was derived. Table 23 above shows the
results of this multivariate analysis using these 11 SNPS and
indicates these SNPs can be analysed in combination to derive a
risk score with clinical utility in discriminating smokers at high
and low risk of lung cancer based on their genotype.
DISCUSSION
[0544] The above results show that several polymorphisms were
associated with either increased or decreased risk of developing
lung cancer. The associations of individual polymorphisms on their
own, while of discriminatory value, are unlikely to offer an
acceptable prediction of disease. However, in combination these
polymorphisms distinguish susceptible subjects from those who are
resistant (for example, between the smokers who develop lung cancer
and those with the least risk with comparable smoking exposure).
The polymorphisms represent exonic polymorphisms known to alter
amino-acid sequence (and likely expression and/or function) in a
number of genes involved in processes known to underlie lung
remodelling and lung cancer, and in one case a silent mutation
having no effect on amino acid composition. The polymorphisms
identified here are found in genes encoding proteins central to
these processes which include inflammation, matrix remodelling,
oxidant stress, DNA repair, cell replication and apoptosis.
[0545] In the comparison of smokers with lung cancer and matched
smokers with near normal lung function (lowest risk for lung cancer
despite smoking), several polymorphisms were identified as being
found in significantly greater or lesser frequency than in the
comparator groups (sometimes including the blood donor cohort). Due
to the small cohort of lung cancer patients, polymorphisms where
there are only trends towards differences (P=0.06-0.25) were
included in the analyses, although in the combined analyses only
those polymorphisms with the most significant differences were
utilised. [0546] In the analysis of the R19W A/G polymorphism of
the Cerberus 1 gene, the AA and AG genotypes were found to be
significantly greater in the lung cancer cohort compared to the
resistant smoker cohort (OR=1.7, P=0.02), consistent with each
having a susceptibility role (see Table 2). The A allele was found
to be significantly greater in the lung cancer cohort compared to
the resistant smoker cohort (OR=1.5, P=0.05), consistent with a
susceptibility role. In contrast, the GG genotype was found to be
greater in the resistant smoker control cohort compared to the lung
cancer cohort, consistent with a protective role (see Table 2).
[0547] In the analysis of the Ser307Ser G/T polymorphism in the
XRCC4 gene, the GG and GT genotypes were found to be greater in the
lung cancer cohort compared to the resistant smoker cohort (OR=1.3,
P=0.12) consistent with each having a susceptibility role. The G
allele was found to be significantly greater in the lung cancer
cohort compared to the resistant smoker controls (OR=1.4, P=0.04),
consistent with a susceptibility role (see Table 3). In contrast,
the TT genotype was found to be greater in the resistant smoker
control compared to the lung cancer cohort, consistent with a
protective role. [0548] In the analysis of the K3326X A/T
polymorphism in the ERCA2 gene, the A/T and TT genotypes were found
to be significantly greater in the lung cancer cohort compared to
the resistant smoker controls (OR=2.5, P=0.04), consistent with a
susceptibility role. The T allele was found to be significantly
greater in the lung cancer cohort compared to the resistant smoker
controls (OR=2.7, P=0.02), see Table 4. In contrast the AA genotype
was found to be greater in the resistant smoker controls compared
to the lung cancer cohort, consistant with a protective role.
[0549] In the analysis of the V433M A/G polymorphism, in the
Integrin alpha-11 gene, the AA genotype was found to be
significantly greater in the lung cancer cohort compared to the
resistant smoker controls (OR=4.3, P=0.002) consistent with a
susceptibility role (see Table 5). The A allele was found to be
significantly greater in the lung cancer cohort compared to the
resistant smoker controls (OR=1.4, P=0.04), consistent with a
susceptibility role (see Table 5). [0550] In the analysis of the
E375G T/C polymorphism in the Calcium/calmodulin-dependent protein
kinase kinase 1 gene, the TT genotype was found to be greater in
the resistant smoker controls compared to the lung cancer cohort
(OR=0.76, P=0.13), consistent with a protective role (see Table 6).
The T allele is found to be greater in resistant smoker controls
compared to the lung cancer cohort (OR=0.84, P=0.14), consistent
with a protective role (see Table 6).
[0551] In the analysis of the -81 C/T (rs 2273953) polymorphism in
the 5' UTR of the gene encoding Tumor protein P73, the CC genotype
was found to be significantly greater in the resistant smoker
cohort compared to the lung cancer cohort (OR=0.46, P<0.001)
consistent with a protective role. The C allele was also found to
be significantly greater in the resistant smoker controls compared
to the lung cancer cohort (OR=0.62, P<0.001), consistent with a
protective role (see Table 7). In contrast, the CT and TT genotypes
were found to be greater in the lung cancer cohort compared to
resistant smoker controls, consistent with a susceptibility
role.
[0552] In the analysis of the A/T c74delA polymorphism in the gene
encoding cytochrome P450 polypeptide CYP3A43, the AT and TT
genotypes were found to be significantly greater in the lung cancer
cohort compared to the resistant smoker cohort (OR=1.74, P=0.05),
consistent with each having a susceptibility role (see Table 8).
The T allele was found to be significantly greater in the lung
cancer cohort compared to the resistant smoker cohort (OR=1.8,
P=0.03), also consistent with a susceptibility role.
[0553] In the analysis of the A/C (rs2279115) polymorphism in the
gene encoding B-cell CLL/lymphoma 2, the AA genotype was found to
be significantly greater in the resistant smoker cohort compared to
the lung cancer cohort (OR=0.69, P=0.05) consistent with a
protective role. The A allele was also found to be significantly
greater in the resistant smoker controls compared to the lung
cancer cohort (OR=0.78, P=0.02), consistent with a protective role
(see Table 9).
[0554] In the analysis of the A/G at +3100 polymorphism in the 3'
UTR (rs2317676) of the gene encoding Integrin beta 3, the AG and GG
genotypes were found to be significantly greater in the resistant
smoker cohort compared to the lung cancer cohort (OR=0.57, P=0.02)
consistent with a protective role. The G allele was also found to
be significantly greater in the resistant smoker controls compared
to the lung cancer cohort (OR=0.54, P=0.01), consistent with a
protective role (see Table 10).
[0555] In the analysis of the -3714 G/T (rs6413429) polymorphism in
the gene encoding Dopamine transporter 1, the TT and GT genotypes
were found to be significantly greater in the lung cancer cohort
compared to the resistant smoker cohort (OR=1.6, P=0.05),
consistent with each having a susceptibility role (see Table
11).
[0556] In the analysis of the A/G (rs1139417) polymorphism in the
gene encoding Tumor necrosis factor receptor 1, the AA genotype was
found to be significantly greater in the lung cancer cohort
compared to the resistant smoker cohort (OR=1.5, P=0.02),
consistent with a susceptibility role (see Table 12). The A allele
was found to be significantly greater in the lung cancer cohort
compared to the resistant smoker cohort (OR=1.3, P=0.04), also
consistent with a susceptibility role.
[0557] In the analysis of the C/Del (rs1799732) polymorphism in the
gene encoding Dopamine receptor D2, the CDel and DelDel genotypes
were found to be significantly greater in the resistant smoker
cohort compared to the lung cancer cohort (OR=0.61, P=0.02)
consistent with each having a protective role. The Del allele was
also found to be significantly greater in the resistant smoker
controls compared to the lung cancer cohort (OR=0.66, P=0.04),
consistent with a protective role (see Table 13).
[0558] In the analysis of the C/T (rs763110) polymorphism in the
gene encoding Fas ligand, the TT genotype was found to be
significantly greater in the resistant smoker cohort compared to
the lung cancer cohort (OR=0.61, P=0.05) consistent with a
protective role (see Table 14).
[0559] In the analysis of the C/T (rs5743836) polymorphism in the
gene encoding Toll-like receptor 9, the CC genotype was found to be
significantly greater in the lung cancer cohort compared to the
resistant smoker cohort (OR=3.1, P=0.02), consistent with a
susceptibility role (see Table 15).
[0560] It is accepted that the disposition to lung cancer is the
result of the combined effects of the individual's genetic makeup
and other factors, including their lifetime exposure to various
aero-pollutants including tobacco smoke. Similarly it is accepted
that lung cancer encompasses several obstructive lung diseases and
characterised by impaired expiratory flow rates (eg FEV1). The data
herein suggest that several genes can contribute to the development
of lung cancer. A number of genetic mutations working in
combination either promoting or protecting the lungs from damage
are likely to be involved in elevated resistance or susceptibility
to lung cancer.
[0561] From the analyses of the individual polymorphisms, 6
protective genotype and 8 susceptibility genotypes were identified
and analysed for their frequencies in the smoker cohort consisting
of resistant smokers and those with lung cancer. A SNP score was
determined for each subject by assigning a score of +1 for the
presence of a susceptibility genotype and -1 for the presence of a
protective genotype. These scores were added to derive a SNP score
for each subject.
[0562] When the frequency of resistant smokers and smokers with
lung cancer were compared according to the SNP score derived from a
5 SNP panel consisting of the SNPs identified in Table 16 herein,
the chances of having lung cancer increased from 24%-31% to 43% in
smokers with a SNP score of -1, 0, or 1+, respectively. When the
frequencies of resistant smokers and smokers with lung cancer were
compared according to a SNP score derived from an 11 SNP panel (11
SNP panel A), it was found that the chances of having lung cancer
increased from 8% to 82% in smokers with a SNP score of 0 compared
to those with a SNP score of 10+.
[0563] A minor increase in the linearity of the relationship
between SNP score and frequency of lung cancer was observed when
the SNP score was derived from a 16 SNP panel consisting of the
SNPs identified in Table 18 herein. Again, the chances of having
lung cancer increased from 8%, to 82% in smokers with a SNP score
of less than or equal to 1 compared to those with a SNP score of
11+. The slight increase in linearity can be seen in a comparison
of FIG. 3 (11 SNP panel B) and FIG. 4 (16 SNP panel).
[0564] When the frequency of resistant smokers and smokers with
lung cancer were compared according to the SNP score derived from a
9 SNP panel consisting of the SNPs identified in Table 21 herein,
the chances of having lung cancer was increased 13-fold in smokers
with a SNP score of 5+compared to those with a SNP score of 1.
[0565] These findings indicate that the methods of the present
invention may be predictive of lung cancer in an individual well
before symptoms present.
[0566] Importantly, a substantial difference is seen in the
distribution of lung cancer patients and control smokers relative
to total SNP score when the SNP score is derived from the 16 SNP
panel rather than from the 11 SNP panel B (see FIG. 8 compared to
FIG. 5). In this analysis, the addition of the 5 SNPs discussed
herein to the 11 SNP panel B results in only a small change to the
linear relationship between lung cancer SNP score and frequency of
lung cancer for the 11 SNP panel B compared to the 16 SNP panel
(see FIGS. 3 and 6, respectively), and results in only a small
difference to the receiver-operator curve analysis with sensitivity
and specificity (see FIGS. 4 and 7, respectively). However, this
addition results in a substantial difference to the utility of the
SNP score, and identifies a larger subgroup of control smokers who
are "low risk" defined by a cut off over the linear scale of SNP
score (see FIG. 8 compared to FIG. 5). A similarly useful
discrimination between lung cancer sufferers and resistant controls
was observed when a distribution of SNP scores calculated using the
9 SNP panel was derived--see FIG. 11. This has important
implications in rationing or prioritising medical
interventions.
[0567] These findings indicate that the methods of the present
invention may be used to identify subsets of nominally at risk
individuals (and particularly smokers) who are at low to average
risk of lung cancer, and are thus not suitable for an
intervention.
[0568] These findings therefore also present opportunities for
therapeutic interventions and/or treatment regimens, as discussed
herein. Briefly, such interventions or regimens can include the
provision to the subject of motivation to implement a lifestyle
change, or therapeutic methods directed at normalising aberrant
gene expression or gene product function. In another example, a
given susceptibility genotype is associated with increased
expression of a gene relative to that observed with the protective
genotype. A suitable therapy in subjects known to possess the
susceptibility genotype is the administration of an agent capable
of reducing expression of the gene, for example using antisense or
RNAi methods. An alternative suitable therapy can be the
administration to such a subject of an inhibitor of the gene
product. In still another example, a susceptibility genotype
present in the promoter of a gene is associated with increased
binding of a repressor protein and decreased transcription of the
gene. A suitable therapy is the administration of an agent capable
of decreasing the level of repressor and/or preventing binding of
the repressor, thereby alleviating its downregulatory effect on
transcription. An alternative therapy can include gene therapy, for
example the introduction of at least one additional copy of the
gene having a reduced affinity for repressor binding (for example,
a gene copy having a protective genotype).
[0569] Suitable methods and agents for use in such therapy are well
known in the art, and are discussed herein.
[0570] The identification of both susceptibility and protective
polymorphisms as described herein also provides the opportunity to
screen candidate compounds to assess their efficacy in methods of
prophylactic and/or therapeutic treatment. Such screening methods
involve identifying which of a range of candidate compounds have
the ability to reverse or counteract a genotypic or phenotypic
effect of a susceptibility polymorphism, or the ability to mimic or
replicate a genotypic or phenotypic effect of a protective
polymorphism.
[0571] Still further, methods for assessing the likely
responsiveness of a subject to an available prophylactic or
therapeutic approach are provided. Such methods have particular
application where the available treatment approach involves
restoring the physiologically active concentration of a product of
an expressed gene from either an excess or deficit to be within a
range which is normal for the age and sex of the subject. In such
cases, the method comprises the detection of the presence or
absence of a susceptibility polymorphism which when present either
upregulates or down-regulates expression of the gene such that a
state of such excess or deficit is the outcome, with those subjects
in which the polymorphism is present being likely responders to
treatment.
Example 5
[0572] This example describes the analysis of the relationship
between SNP score and risk of the four most common types of lung
cancer.
[0573] The lung cancer cohort described in Example 1 above is
typical of that seen in other reported lung cancer studies. In
particular, the distribution of the four leading histological types
of primary lung cancer is consistent with larger studies. Here, 45%
of subjects had adenocarcinoma, 23% of subjects had squamous cell
lung cancer, 16% of subjects had small cell lung cancer, and 13% of
subjects had non-small cell lung cancer.
[0574] Reporters of epidemiological studies have suggested that
smoking plays a greater role in small cell and squamous cell lung
cancer and less in adenocarcinoma. The basis of this suggestion is
not certain. The role of genetic factors in each histological type
of lung cancer is unknown.
[0575] When the relationship between SNP score (determined as
described above) and risk of lung cancer was examined according to
histological type, the risk (Odds ratio) is higher for those with
small-cell lung cancer and squamous cell lung cancer while least
for those with adenocarcinoma (see FIG. 12).
[0576] Without wishing to be bound by any theory, this suggests
that the genetic effect measured by the SNP score may interact with
smoking to confer risk of lung cancer. It also suggests, again
without wishing to be bound by any theory, that the SNP score
effect, although present, is least for lung cancer of the
adenocarcinoma type (typically seen in light smokers or
non-smokers). Collectively this example shows that the SNP score
has utility in identifying those at risk of all types of lung
cancer, and that an analysis of SNP score may be useful in
determining not only whether or not an intervention in respect of a
subject is warranted or desirable, but also the type of
intervention. For example, on the basis of their SNP score, a
subject may be considered suitable for more frequent screening
(e.g., for rapidly-growing or aggressive lung cancer types).
Example 6
[0577] This example presents the identification and analysis of a
19 SNP panel (11 susceptibility SNPs) and 8 protective SNPs as
shown in Table 24 below useful for the methods of the present
invention.
[0578] Statistical Analysis
[0579] Patient characteristics in the lung cancer sufferers and
controls were compared by unpaired t-tests for continuous variables
and chi-square test or Fisher's exact test for discrete variables.
Genotype and allele frequencies were checked for Hardy Weinberg
Equilibrium and population admixture by the Population structure
analysis by genotyping 40 unrelated SNPs. Distortions in the
genotype frequencies between lung cancer sufferers and controls
were identified using 2 by 3 contingency tables. Where the
homozygote genotype (recessive model) or combined homozygote and
heterozygote genotypes (codominant model) for the minor allele were
found in excess in the healthy smokers controls compared to the
lung cancer cohort, these SNP genotypes were assigned as
protective. Where the homozygote genotype (recessive model) or
combined homozygote and heterozygote genotypes (codominant model)
for the minor allele were found in excess in the lung cancer cohort
compared to healthy smokers controls, these SNP genotypes were
assigned as susceptible. The magnitude of the effect from each SNP
was analysed using univariate analysis and multivariate analysis.
Based on these analyses, SNPs were ranked according to their
ability to discriminate between lung cancer sufferers and controls,
and combined as described to generate the SNP score. Non-genetic
risk factors including age and family history were also analysed,
and combined with the SNP score to generate a composite SNP
score.
[0580] Results
[0581] Table 24 below summarises the univariate analysis showing
protective and susceptibility SNPs associated with lung cancer as
set out herein. Odd's ratios (OR) and p values are for cancer
patients compared to resistant smokers with normal lung function.
Table 24 also summarises the multivariate analysis, where stepwise
regression analysis was performed and chi squared values are
presented for each polymorphism.
TABLE-US-00028 TABLE 24 Genotypes and results of regression
analysis - 19 SNP panel Lung Smoking Call Univariate Multivariate
SNP (rs#) Genotype cancer controls rate OR P value Point estimate P
value Phenotype CYP 2E1 TT/TC 24 (6%) 14 (3%) 95% 2.1 0.03 0.63
0.24 susceptibility (Rsa 1 C/T) CC 379 (94%) 463 (97%) (1.0-4.3)
(0.29-1.37) Interleukin-18 CC 237 (54%) 208 (45%) 96% 1.4 0.009
0.65 0.007 susceptibility (-133 G/C) CG/GG 201 (46%) 250 (55%)
(1.1-1.9) (0.48-0.89) Interleukin-8 TT 129 (31%) 109 (23%) 96% 1.5
0.005 0.72 0.06 susceptibility (-251 A/T) AT/AA 284 (69%) 367 (77%)
(1.1-2.1) (0.51-1.02) Interleukin 1B GG 215 (49%) 212 (44%) 99% 1.2
0.14 0.86 0.33 susceptibility (rs 16944) AA/AG 224 (51%) 269 (56%)
(0.9-1.6) (0.63-1.17) ITGA11 AA 14 (3%) 6 (1%) 98% 2.6 0.04 0.28
0.02 susceptibility (rs2306022) GA/GG 422 (97%) 470 (99%) (0.9-7.6)
(0.10-0.84) N-acetylcysteine GG 239 (56%) 222 (47%) 97% 1.4 0.006
0.76 0.08 susceptibility transferase 2 AA/AG 189 (44%) 253 (53%)
(1.1-1.9) (0.56-1.03) (rs 1799930) .alpha.1-antichymotrypsin GG 123
(28%) 96 (20%) 98% 1.6 0.004 0.69 0.05 susceptibility (-15 A/G)
AG/AA 312 (72%) 383 (80%) (1.2-2.2) (0.48-0.99) Cerberus 1 AA/AG 71
(16%) 59 (12%) 97% 1.4 0.10 0.71 0.10 susceptibility (rs 10115703)
GG 363 (84%) 413 (88%) (0.9-2.0) 0.45-1.10 DAT1 GT/TT 64 (15%) 50
(10%) 98% 1.5 0.04 0.68 0.06 susceptibility (rs6413429) GG 367
(85%) 431 (90%) (1.0-2.3) (0.43-1.10) TNFR1 AA 148 (36%) 142 (30%)
96% 1.3 0.05 0.88 0.20 susceptibility (rs1139417) AG/GG 258 (64%)
329 (70%) (1.0-1.8) (0.64-1.23) TLR9 CC 12 (3%) 6 (1%) 96% 2.2 0.12
0.57 0.33 susceptibility (rs5743836) CT/TT 419 (97%) 455 (99%)
(0.8-6.6) (0.19-1.75) P73 CC 219 (52%) 292 (62%) 96% 0.65 0.001
1.50 0.01 protective (rs 2273953) TC/TT 206 (48%) 178 (38%)
(0.49-0.85) ( 1.1-2.04) SOD3 GG/GC 4 (1%) 15 (3%) 96% 0.28 0.02
8.43 0.01 protective (rs1799895) CC 425 (99%) 451 (97%) (0.10-0.90)
(1.65-43.22) ITGB3 GG/GA 44 (10%) 77 (16%) 98% 0.59 0.008 1.4 0.009
protective (rs2317676) AA 391 (90%) 403 (84%) (0.39-0.89)
(1.17-3.00) DRD2 CDel/Del.Del 70 (16%) 107 (22%) 98% 0.68 0.02 1.80
0.005 protective (rs 1799732) CC 359 (84%) 372 (78%) (0.48-0.96)
(1.20-2.70) BCL2 AA 103 (24%) 145 (31%) 97% 0.71 0.03 1.4 0.05
protective (rs 2279115) AC/CC 328 (76%) 330 69%) (0.53-0.97)
(1.01-2.04) XPD GG 60 (14%) 81 (18%) 96% 0.74 0.11 1.35 0.18
protective (rs 13181) GT/TT 376 (86%) 377 (82%) (0.51-1.10)
(0.90-2.10) REV1 CC 128 (29%) 163 (34%) 98% 0.79 0.10 1.34 0.08
protective (rs3087386) TC/TT 310 (71%) 312 (66%) (0.59-1.10)
(0.97-1.87) FasL TT 53 (12%) 78 (16%) 98% 0.72 0.09 1.46 0.10
protective (rs763110) TC/CC 379 (88%) 403 (84%) (0.49-1.10)
(0.93-2.29)
[0582] Having defined the SNP panel SNP score, the genetic data was
then analysed together with non-genetic data (specifically age,
family history, history of COPD, and smoking exposure). Using
multiple regression analysis, the magnitude of the effect of the 19
SNP panel in relation to age, family history and smoking exposure
was determined. A score for age (+4 for those over 60 years old),
history of COPD (+4 for those with self reported COPD/emphysema)
and family history (+3 to those with a first degree relative with
lung cancer) was then assigned. As smoking exposure was a
recruitment criteria, only a small contribution from smoking
exposure was observed and was thus omitted from the composite SNP
score. This SNP score was compared with (a) the frequency of lung
cancer, and (b) the floating absolute relative risk among the
combined smoking cohort.
[0583] A linear relationship was observed across composite lung
cancer SNP scores .ltoreq.1 to 8+ with lung cancer frequency
spanning 15% to 85% (FIG. 13a). The magnitude of the effect was
examined using the floating absolute risk plotted on a log scale
(equivalent to an Odds ratio, OR), which references the lowest
frequency group as 1 (referent group, lung cancer score .ltoreq.1)
and compares each lung cancer score relative to the referent group
(FIG. 13b). The OR ranged from 1 to 31.5 across the lung cancer
scores when subjects are grouped roughly as quintiles. The OR was
even higher for those with a SNP score of 9+.
[0584] In a receiver operator curve analysis, the area under the
curve (AUC, or C statistic) for the 19 SNP panel, age, family
history of lung cancer, and history of COPD were 0.68, 0.70, 0.55,
and 0.62, respectively. The distribution of the SNP score between
cases and controls for the total cohort (n=930) shows a bimodal
distribution (FIG. 14a). Corresponding sensitivities and
specificities on receiver-operator-curve analyses are shown in
Table 25 below.
TABLE-US-00029 TABLE 25 Sensitivity and specificity estimates - 19
SNP panel Lung cancer score Sensitivity 95% CI Specificity 95% CI
.gtoreq.1 95% 94-98% 23% 19-27% .gtoreq.3 89% 86-92% 44% 39-48%
.gtoreq.7 50% 45-55% 89% 86-91% .gtoreq.9 28% 23-32% 98% 96-99%
[0585] Discussion
[0586] The composite SNP score derived from the 19 SNP panel in
combination with non-genetic risk factores as described in this
example generated a C statistic of 0.78, and a cut off of .gtoreq.3
with a sensitivity of 89% and corresponding specificity of 44%.
[0587] The C statistic for the SNP score derived from the 19 SNP
panel in the absence of non-genetic risk factors was 0.70,
indicating its useful predictive and discriminatory utility and
suitability for use in the methods described herein, both on its
own or in combination with non-genetic risk factors.
Example 7
[0588] Table 26 below presents representative examples of
polymorphisms in linkage disequilibrium with the polymorphisms
specified herein. Examples of such polymorphisms can be located
using public databases, such as that available at www.hap,ap.org.
Specified polymorphisms are shown in parentheses. The rs numbers
provided are identifiers unique to each polymorphism.
TABLE-US-00030 TABLE 26 Polymorphism reported to be in LD with
polymorphisms specified herein. CAMKK1 rs11078470 rs1029801
rs11650638 rs1029800 (rs7214723) rs6502751 rs7214864 rs9914305
rs2058257 rs8065798 rs9904678 rs7223713 rs4790546 rs7208983
rs9898774 rs7223709 rs7212114 rs11651131 rs7221812 rs12150410
rs7221971 rs9897177 ITGA11 rs11633421 rs6494734 rs898581 rs1239019
rs964691 rs898580 rs3736495 rs8025985 rs11072008 rs3736494
rs2306025 rs12050550 rs3736493 rs2306024 rs716379 rs8041788
rs2306023 rs1380883 rs8043152 (rs2306022) rs3784342 rs16951774
rs898586 rs1380882 rs1996361 rs12442156 rs3784344 rs5016065
rs7176011 rs3784345 rs2899735 rs7176339 rs11632266 rs2414996
rs898585 rs1124577 rs2414997 rs4776395 rs7177709 rs7171871
rs7182350 rs3784346 rs1516869 rs12908869 rs7180218 rs16951777
rs7161871 rs748891 rs16951778 rs11632400 rs748892 rs3784335
rs898584 rs17266192 rs17318470 rs16951816 rs898579 rs3784336
rs7179347 rs12440936 rs3784337 rs7178537 rs748971 rs16951779
rs7179545 rs8029838 rs898588 rs2125998 rs16951835 rs7163918
rs10162690 rs8031003 rs2271723 rs9302249 rs4776396 rs898587
rs7162991 rs2306021 rs1237911 rs6494735 rs16951841 rs2271722
rs4777040 rs11072006 rs6494736 rs11630928 rs8030178 rs11635643
rs8029230 rs4777037 rs8028967 rs3736491 rs8028971 rs7176267
rs8029113 rs11072007 rs4777041 rs4777038 rs8029452 rs4777039
rs7169899 rs1533469 rs4777042 rs11858293 rs8035990 rs7179228
rs2414998 rs7179598 rs16951819 rs8042664 rs2169214 rs11852504
rs12912832 rs7167822 rs2125997 rs2292745 rs7181259 rs7168069
rs1975874 rs7169698 rs898583 rs6494733 rs16951820 rs970264 rs898582
rs1319223 rs1563894 CER1 rs10810224 rs17289263 rs3761666 rs13286013
rs7022304 rs7870750 rs10961679 rs7022400 rs10121506 rs10961680
rs11999277 rs10118242 rs10961681 rs1494360 rs10118290 rs951273
rs1494359 rs16932212 rs2131883 rs1494358 rs11794846 rs2131882
rs1494357 rs10122395 rs12338263 rs3747532 rs10125285 rs12338303
(rs10115703) rs1494351 rs12338380 rs10122490 rs1494350 rs2088042
rs7018937 rs10961683 rs12347640 rs12115314 rs10961684 rs10122817
rs7035643 rs11793334 rs12115487 rs10961682 rs7019731 rs11789968
rs7019387 rs10810225 rs3761665 rs3819004 rs10123442 rs7036635
rs10810226 XRCC4 rs36059813 rs28360323 rs10514256 rs35770549
rs28360322 rs10514255 rs35770061 rs28360321 rs10514254 rs35704249
rs28360320 rs10434637 rs35694031 rs17567561 rs10078343 rs35618200
rs17205881 rs10070866 rs35262280 rs16900371 rs10067830 rs35219614
rs16900367 rs10061326 rs35211331 rs16900363 rs10061086 rs34801422
rs16900362 rs10057194 rs34697956 rs16900361 rs10057054 rs34646294
rs16900359 rs9293337 rs34626079 rs16900357 rs9293336 rs34544738
rs16900353 rs9293335 rs34326210 rs16900343 rs7736592 rs34164901
rs16900342 rs7735781 rs34052855 rs16900341 rs7734849 rs34006354
rs16900340 rs7729473 rs28746479 rs16900339 rs7729020 rs28746478
rs16900330 rs7728486 rs28746477 rs16900328 rs7727606 rs28746476
rs16900325 rs7716696 rs28360351 rs16900322 rs7714809 rs28360350
rs16900317 rs7711016 rs28360349 rs16900315 rs6869679 rs28360348
rs13359237 rs4987240 rs28360347 rs13358544 rs4703951 rs28360346
rs13357939 rs4703950 rs28360345 rs13187520 rs4703568 rs28360344
rs13167490 rs4438854 rs28360343 rs13167223 rs3910950 rs28360342
rs13163691 rs3836874 rs28360341 rs13163534 rs3836873 rs28360340
rs13155538 rs3777020 rs28360339 rs12697728 rs3777019 rs28360338
rs12520831 rs3777018 rs28360337 rs12186876 rs3777015 rs28360336
rs11960030 rs2891980 rs28360335 rs11960003 rs2386275 rs28360334
rs11959198 rs2084099 rs28360333 rs11958342 rs2035990 rs28360332
rs11955413 rs1805377 rs28360331 rs11954157 (rs1056503) rs28360330
rs11953364 rs382069 rs28360329 rs11950724 rs301292 rs28360328
rs11749552 rs301291 rs28360327 rs10805813 rs177712 rs28360326
rs10805812 rs28360325 rs10642662 rs28360324 rs10514257 BRCA2
rs36116910 rs28897730 rs11571808 rs11571701 rs11571598 rs7337784
rs773032 rs36114000 rs28897729 rs11571807 rs11571700 rs11571597
rs7337574 rs773031 rs36091054 rs28897728 rs11571806 rs11571699
rs11571596 rs7337016 rs773030 rs36073425 rs28897727 rs11571805
rs11571698 rs11571595 rs7336403 rs773029 rs36060526 rs28897726
rs11571804 rs11571697 rs11571594 rs7334543 rs773027 rs36018961
rs28897725 rs11571803 rs11571696 rs11571593 rs7332492 rs766173
rs35979864 rs28897724 rs11571802 rs11571695 rs11571592 rs7331638
rs721185 rs35930474 rs28897723 rs11571801 rs11571694 rs11571591
rs7330025 rs703224 rs35768834 rs28897722 rs11571800 rs11571693
rs11571590 rs7328654 rs703223 rs35697303 rs28897721 rs11571799
rs11571692 rs11571589 rs7328264 rs703213 rs35685866 rs28897720
rs11571798 rs11571691 rs11571588 rs7328101 rs693963 rs35628833
rs28897719 rs11571797 rs11571690 rs11571587 rs7327867 rs664345
rs35596121 rs28897718 rs11571796 rs11571689 rs11571586 rs7327813
rs651906 rs35573139 rs28897717 rs11571794 rs11571688 rs11571585
rs7327677 rs573014 rs35571300 rs28897716 rs11571792 rs11571687
rs11571584 rs7327471 rs559067 rs35563967 rs28897715 rs11571791
rs11571686 rs11571583 rs7324145 rs543304 rs35527903 rs28897714
rs11571790 rs11571685 rs11571582 rs7320990 rs542551 rs35497963
rs28897713 rs11571789 rs11571684 rs11571581 rs7318434 rs517118
rs35486082 rs28897712 rs11571788 rs11571683 rs11571580 rs6561306
rs472817 rs35477961 rs28897711 rs11571787 rs11571682 rs11571579
rs5802644 rs396579 rs35408951 rs28897710 rs11571786 rs11571681
rs11571578 rs4987117 rs206346 rs35382259 rs28897709 rs11571784
rs11571680 rs11571577 rs4987049 rs206344 rs35335654 rs28897708
rs11571782 rs11571679 rs11571576 rs4987048 rs206343 rs35324259
rs28897707 rs11571780 rs11571678 rs11571575 rs4987047 rs206342
rs35315530 rs28897706 rs11571779 rs11571676 rs11571574 rs4987046
rs206341 rs35188168 rs28897705 rs11571778 rs11571675 rs11552891
rs4986860 rs206340 rs35069894 rs28897704 rs11571777 rs11571674
rs11464335 rs4986859 rs206319 rs35029074 rs28897703 rs11571776
rs11571673 rs11460904 rs4986858 rs206318 rs35027705 rs28897702
rs11571775 rs11571672 rs11451886 rs4986856 rs206147 rs35005399
rs28897701 rs11571774 rs11571671 rs11426352 rs4942505 rs206146
rs34959007 rs28897700 rs11571773 rs11571670 rs11371521 rs4942499
rs206145 rs34943677 rs28657708 rs11571772 rs11571669 rs11327981
rs4942486 rs206123 rs34926095 rs28641896 rs11571771 rs11571668
rs11312202 rs4942485 rs206122 rs34925070 rs28569916 rs11571770
rs11571667 rs11306457 rs4942448 rs206121 rs34895626 rs28479757
rs11571769 rs11571666 rs11291838 rs4942443 rs206120 rs34891002
rs28473213 rs11571768 rs11571665 rs11147494 rs4942440 rs206099
rs34842101 rs17692629 rs11571767 rs11571664 rs11147493 rs4942439
rs206098 rs34841049 rs17636116 rs11571766 rs11571663 rs11147492
rs4942423 rs206097 rs34835575 rs17077554 rs11571765 rs11571662
rs11147491 rs4570704 rs206096 rs34816981 rs17077542 rs11571764
rs11571661 rs11147490 rs3837580 rs206095 rs34809891 rs17077541
rs11571763 rs11571660 rs11147489 rs3803282 rs206081 rs34770647
rs17077519 rs11571762 rs11571659 rs11147488 rs3783265 rs206080
rs34704662 rs13378910 rs11571761 rs11571658 rs11147486 rs3764792
rs206079 rs34692639 rs13378905 rs11571760 rs11571657 rs10870659
rs3764791 rs206078 rs34647461 rs13378423 rs11571759 rs11571656
rs10577567 rs3752451 rs206077 rs34578379 rs13378422 rs11571758
rs11571655 rs10492397 rs3752448 rs206076 rs34578349 rs12871316
rs11571757 rs11571654 rs10492396 rs3752447 rs206075 rs34575057
rs12871310 rs11571756 rs11571653 rs10492395 rs3752446 rs206074
rs34469166 rs12869544 rs11571754 rs11571652 rs9943890 rs3210648
rs206073 rs34437679 rs12869093 rs11571753 rs11571651 rs9943888
rs3092990 rs206072 rs34380010 rs12868315 rs11571752 rs11571650
rs9943876 rs3072043 rs206071 rs34370449 rs12862392 rs11571751
rs11571649 rs9634798 rs3072042 rs206070 rs34355306 rs12862064
rs11571750 rs11571648 rs9634797 rs3072040 rs206069 rs34351119
rs12862049 rs11571749 rs11571647 rs9634796 rs2761367 rs206068
rs34345002 rs12859126 rs11571748 rs11571646 rs9634672 rs2761363
rs206067 rs34309943 rs12859094 rs11571747 rs11571644 rs9595469
rs2320236 rs189979 rs34288419 rs12859079 rs11571746 rs11571643
rs9595468 rs2238163 rs176176 rs34273171 rs12858763 rs11571745
rs11571642 rs9595456 rs2238162 rs169548 rs34225677 rs12858735
rs11571744 rs11571641 rs9595402 rs2227944 rs169547 rs34184533
rs12858723 rs11571743 rs11571640 rs9595395 rs2227943 rs169546
rs34178365 rs12858361 rs11571742 rs11571639 rs9590958 rs2219594
rs144848 rs34175773 rs12854843 rs11571741 rs11571638 rs9590951
rs2126042 rs15869 rs34108667 rs12853807 rs11571740 rs11571637
rs9590940 rs2100785 rs34102917 rs12561064 rs11571739 rs11571636
rs9590939 rs1963505 rs34080444 rs12429216 rs11571738 rs11571635
rs9590938 rs1853521 rs34075550 rs12017223 rs11571737 rs11571634
rs9567674 rs1853520 rs34009686 rs11842816 rs11571736 rs11571633
rs9567670 rs1853519 rs34001953 rs11841349 rs11571735 rs11571632
rs9567666 rs1801499 rs28897762 rs11839855 rs11571734 rs11571631
rs9567654 rs1801439 rs28897761 rs11620336 rs11571733 rs11571630
rs9567639 rs1801426 rs28897760 rs11616673 rs11571732 rs11571629
rs9567623 rs1801406 rs28897759 rs11571837 rs11571731 rs11571628
rs9567609 rs1799968 rs28897758 rs11571836 rs11571730 rs11571627
rs9567605 rs1799956 rs28897757 rs11571835 rs11571729 rs11571626
rs9567600 rs1799955 rs28897756 rs11571834 rs11571728 rs11571625
rs9567582 rs1799954 rs28897755 (rs11571833) rs11571727 rs11571624
rs9567578 rs1799953 rs28897754 rs11571832 rs11571726 rs11571623
rs9567576 rs1799952 rs28897753 rs11571831 rs11571725 rs11571622
rs9551726 rs1799951 rs28897752 rs11571830 rs11571723 rs11571621
rs9534367 rs1799944 rs28897751 rs11571829 rs11571722 rs11571620
rs9534344 rs1475990 rs28897750 rs11571828 rs11571721 rs11571619
rs9534342 rs1460817 rs28897749 rs11571827 rs11571720 rs11571618
rs9534323 rs1460816 rs28897748 rs11571826 rs11571719 rs11571617
rs9534318 rs1380946 rs28897747 rs11571825 rs11571718 rs11571616
rs9534286 rs1207954 rs28897746 rs11571824 rs11571717 rs11571615
rs9534275 rs1207953 rs28897745 rs11571823 rs11571716 rs11571614
rs9534274 rs1207952 rs28897744 rs11571822 rs11571715 rs11571613
rs9534270 rs1148321 rs28897743 rs11571821 rs11571714 rs11571612
rs9534269 rs1148320 rs28897742 rs11571820 rs11571713 rs11571611
rs9534268 rs1128611 rs28897741 rs11571819 rs11571712 rs11571610
rs9534262 rs1128610 rs28897740 rs11571818 rs11571711 rs11571609
rs9534259 rs1062947 rs28897739 rs11571817 rs11571710 rs11571608
rs9534174 rs1062946 rs28897738 rs11571816 rs11571709 rs11571607
rs9526165 rs1046984 rs28897737 rs11571815 rs11571708 rs11571606
rs9526160 rs1045789 rs28897736 rs11571814 rs11571707 rs11571605
rs9526148 rs1029304 rs28897735 rs11571813 rs11571706 rs11571604
rs9526131 rs1012130 rs28897734 rs11571812 rs11571705 rs11571603
rs7992196 rs1012129 rs28897733 rs11571811 rs11571704 rs11571602
rs7982943 rs811637 rs28897732 rs11571810 rs11571703 rs11571601
rs7981512 rs798652 rs28897731 rs11571809 rs11571702 rs11571600
rs7491644 rs773033 P73 rs3765702 rs1122638 rs3819955 rs5031051
rs3753205 rs3765703 rs12062249 rs3765707 rs5031052 rs3765714
rs10910007 rs2368542 rs12059298 (rs2273953) rs3765715 rs12028205
rs6665164 rs3765708 rs1801173 rs3765716 rs12057230 rs7554226
rs12025725 rs4648547 rs1122723 rs12024891 rs10910009 rs3765709
rs1122724 rs10910008 rs1885874 rs3765710 rs1122725 rs12121199
rs12403618 rs3765711 rs12095743 rs3765705 rs12403927 rs3765712
rs1122639 rs3765706 rs10910010 rs3765713 CYP3A43 rs2738258
rs1041966 rs2023548 rs688926 rs13236744 rs2687110 rs12721632
rs493380 rs687134 rs13444455 rs17294659 rs12721636 rs1554511
rs4236544 rs10241225 rs12670850 rs12721633 rs667660 rs4646472
rs10225908 rs6970689 rs12721637 rs620020 rs671673 rs2897018
rs11768200 rs1800713 rs17161937 rs660629 rs528144 rs6465753
rs2740574 rs1403195 rs533486 rs6975773 rs4986914 rs473706 rs501275
rs2263430 rs2740573 rs10255255 rs641815 rs6415332 rs11773597
rs585071 rs641761 rs2263431 rs1851426 rs2023165 rs472667 rs2687106
rs12114000 rs1036374 rs579424 rs10270146 rs2740572 rs651430
rs13234698 rs12721619 rs4301384 rs653245 rs549061 rs12721625
rs2740571 rs7807561 rs545400 rs3800957 rs2687103 rs800675 rs4646474
rs7801671 rs7811022 rs558112 rs487813 rs16867648 rs7811025 rs558002
rs1077078 rs2687105 rs2740570 (C74 delA) rs679320 rs2687104
rs4729550 rs13236405 rs678040 rs10264769 rs3958412 rs800674
rs568859 rs2405184 rs1320390 rs800673 rs800667 rs2740575 rs1320389
rs523407 rs6960775 rs2253498 rs2687102 rs642761 rs565079 rs2253493
rs2687101 rs496000 rs675644 rs17161904 rs2740569 rs800672 rs648515
rs4602816 rs2687100 rs4268042 rs800666 rs3991692 rs2737418 rs892753
rs646563 rs6957392 rs760368 rs12671336 rs694939 rs12721634
rs2017121 rs2164226 rs800664 BCL2 rs12458289 rs1473418 rs2551407
rs2849372 rs949037 (rs2279115) rs10460159 rs2615196 rs2849380
rs2551400 rs2849383 rs2849371 rs1462128 rs2551401 rs11663788
rs8098151 rs1462129 rs7243985 rs2849367 rs3786327 rs2051424
rs2551402 rs6810 rs2850757 rs2051423 rs8099294 rs2615201 rs2850756
rs1944422 rs2051422 rs736223 rs2551410 rs2085958 rs1944423 rs898891
rs1893805 rs12455492 rs11659773 rs2850767 rs2032343 rs7239542
rs2551403 rs2850768 rs11152379 rs1541295 rs2551404 rs2551408
rs1541296 rs4987712 rs11660715 rs2236719 rs1809319 rs1893806
rs17687494 rs2849376 rs2003149 rs4987711 rs8094041 rs2849375
rs439670 rs4987710 rs2551405 rs12327344 rs489520 rs1800477
rs2850764 rs2255302 rs3744939 rs1801018 rs10460158 rs12953721
rs428356 rs4987707 rs2551406 rs8083276 rs383770 rs4987706 rs698708
rs7231949 ITGB3 rs884696 rs8074348 rs951351 rs13380810 rs9303533
rs7219925 rs16941796 rs7218632 rs7223956 rs7214993 rs10514919
rs2015729 rs11651736 rs7220606 rs8075031 rs11870334 rs16941801
rs3785870 rs12162128 rs1051452 rs11870365 rs7217214 rs16941829
rs7224753 rs16941864 rs16941776 rs16941802 rs2292864 rs7221196
(rs2317676) rs16941780 rs7212751 rs12940355 rs12603582 rs3809865
rs11657517 rs11649785 rs12951133 rs12603725 rs9916007 rs11658221
rs1000232 rs12942670 rs10221263 rs8068200 rs8073827 rs2292866
rs12943780 rs12602240 rs9894860 rs12941431 rs2292867 rs12942968
rs11870252 rs12600603 rs11651758 rs16941807 rs12951679 rs11867253
rs9893410 rs12453200 rs11079770 rs12942997 rs11867192 rs7209109
rs11651904 rs8073229 rs12943005 rs3785873 rs7225700 rs11656865
rs11868912 rs13306482 rs9747605 rs4968313 rs7503748 rs1878067
rs1969268 rs9906248 rs2317677 rs11657963 rs988684 rs1533409
rs3760372 rs11658426 rs984370 rs5918 rs15908 rs13306488 rs11650072
rs8080254 rs5920 rs12709459 rs13306489 rs11079772 rs8074094
rs13306485 rs13306483 rs1969267 rs12600865 rs8066295 rs12709458
rs2292863 rs8081202 rs4968314 rs11870620 rs13306486 rs5921
rs16941855 rs7218813 rs11079769 rs5917 rs4642 rs9914944 rs9899121
rs4486970 rs2292699 rs13306487 rs11869835 rs10853089 rs3851806
rs2292700 rs4634 rs12950632 rs6504833 rs16941793 rs8064853
rs7214096 rs3744452 rs7209700 rs9912177 rs7217710
rs3744453 rs4968312 rs13306484 rs7214468 rs11868344 rs8078614
rs12451759 rs11656809 rs11870781 rs3851807 rs5919 rs999323
rs16941861 rs12940207 rs13306476 rs3785872 rs3809863 rs8064871
rs13306477 rs12949936 rs11655943 rs8069732 rs13306478 rs11079771
rs16941863 rs11868894 rs2292865 rs11650022 rs9674670 rs8077753
rs13306480 rs7211018 rs9284377 DAT1 rs2937639 rs2447848 rs11564751
rs2617592 rs1354139 rs2550961 rs2447847 rs4029364 rs2617591
rs2652505 rs2550962 rs2516289 rs4029363 rs2652508 rs11747778
rs11564757 rs2617601 rs2937637 rs2617590 rs2078247 rs2550963
rs2735855 rs2937636 rs2617589 rs2617584 rs2937638 rs3776485
rs7733388 rs2471921 rs2550939 rs1316830 rs2550967 rs11564750
rs2617588 rs2113330 rs2735859 rs2617600 rs2550956 rs2550949
rs2975224 rs2735858 rs2735854 rs11564749 rs2652506 rs2617583
rs2859604 rs2735935 rs2652510 rs10070282 rs12652860 rs2550965
rs2735934 rs2937635 rs10079467 rs12654851 rs2516291 rs2617599
rs2975225 rs2550947 rs6879432 rs2447850 rs2975227 rs3756450
rs2550946 rs9312868 rs2516290 rs2975226 rs2617595 rs2550945
rs1478435 rs2550966 rs2652513 rs2652509 rs2550944 rs1478434
rs2254255 rs2652512 rs2617594 rs250694 rs10063727 rs2963238
rs456323 rs2550955 rs2550943 rs4639276 rs2617603 rs2617598
rs2550954 rs565988 rs2911493 rs2735853 rs2550953 rs565985 rs2471926
rs2735852 rs2550952 rs250693 rs2447849 rs2617597 rs2550951
rs2550941 rs2617602 rs2652511 rs2550950 rs250692 rs11564752
rs2617596 rs2963236 rs193941 rs2735857 rs2550957 rs2617593 rs565123
rs2735856 (rs6413429) rs193942 rs2550940 TNFR1 rs1800693 rs4149636
rs4149581 rs4149625 rs4149618 rs4149642 rs2363888 rs4149580
rs4149571 rs4149617 rs4149641 rs4149635 rs4149579 rs4149624
rs12300705 rs4149587 rs877249 rs4149578 rs4149623 rs11064143
rs4149640 rs4149583 rs4149577 rs4149622 rs7297961 rs12832171
rs4149634 rs4149627 rs4441073 rs11064145 rs11525582 rs2284344
rs4149626 rs767455 rs11608320 rs4149586 rs4149633 rs10774425
(rs1139417) rs11608322 rs4149639 rs4149632 rs11836766 rs2234649
rs2228576 rs12317730 rs4149631 rs4149576 rs4149621 rs1800692
rs4149630 rs4149575 rs4149570 rs4149638 rs887477 rs4149574
rs16932532 rs4149585 rs4149629 rs4149573 rs4149620 rs4149584
rs4149582 rs4149572 rs4149619 rs4149637 rs1860545 rs11615387
rs4149569 DRD2 rs17529477 rs4337071 rs5013062 rs12099213 rs12361261
rs17601612 rs4630328 rs12364283 rs7934294 rs4466875 rs11214610
rs11214612 rs11214617 rs12574578 rs7131411 rs4245146 rs11214613
rs7110440 rs11301285 rs4429089 rs4245147 rs11214614 rs12808668
rs17602285 rs4245153 rs4936270 rs4350392 rs12785817 rs11214627
rs4245154 rs4936271 rs11601054 rs4483623 rs10891564 rs11214636
rs4936272 rs7930567 rs10891556 rs6589379 rs4938026 rs4274224
rs12225915 rs4424703 rs4938023 rs2002229 rs4245148 rs10891553
rs11214618 rs4503578 rs2002228 rs4460839 rs12421616 rs12800185
rs4254099 rs12280961 rs12576411 rs4245149 rs7121986 rs7111031
rs12291458 rs7109897 rs7102650 rs6589377 rs4938024 rs2514218
rs17115596 rs7939472 rs11214619 rs6589381 rs2511514 rs12805897
rs11214615 rs4482060 rs6589382 rs11214642 rs4581480 rs4938019
rs10891562 rs4245151 rs7122454 rs12417718 rs4421776 rs7949802
rs7948028 rs11214616 rs11214623 rs11214633 rs10891550 rs10891554
rs4611239 rs11214634 rs7131056 rs4533070 rs4245150 rs12275979
rs11214611 rs10789943 rs17602038 rs4938025 rs4936274 rs3935565
rs4938021 rs7928940 rs12291794 rs10789944 rs4936275 rs4479021
rs4648317 rs7116768 rs4936276 rs12418281 rs7109615 rs12281924
rs1986665 rs7479729 rs10891551 (rs1799732) rs12363546 rs7106947
rs4322431 rs1799978 rs12576181 rs4447205 rs7117915 rs5013059
rs10736466 rs1984739 rs10891552 rs5013060 rs4938022 rs4245152
rs7118174 rs5013061 rs12292637 rs4534613 FasL rs1894626 rs2859235
rs2639617 rs3021335 rs16844867 rs639622 rs10912122 rs2859239
rs2933547 rs9787393 rs2639621 rs2639618 rs2639616 rs2859244
rs9787248 rs2859228 rs2859236 rs2131373 rs2859245 rs12080307
rs2859229 rs10798130 rs12130118 rs10753023 rs749154 rs1492899
rs16844856 rs2859240 rs10798133 rs749155 rs12082528 rs2021839
rs2639615 rs2859246 (rs763110) rs4304626 rs2021838 rs2859241
rs2859247 rs2859233 rs2859237 rs2859242 rs2639614 rs2859234
rs2859238 rs2859243 rs2859248 TLR9 rs353551 rs352168 rs17052020
rs5743847 rs5743838 rs352158 rs352167 rs10212560 rs445676 rs5743837
rs614288 rs352166 rs12629425 rs5743846 (rs5743836) rs6767333
rs13064414 rs9816466 rs5743845 rs187084 rs9828488 rs352165
rs7614535 rs352140 rs352173 rs11712164 rs352162 rs5743844 rs352172
rs17052017 rs5743850 rs5743843 rs3774412 rs9813448 rs6809796
rs5743842 rs709315 rs9813468 rs13080616 rs352139 rs352171 rs352164
rs13060808 rs5743841 rs352170 rs352163 rs5743849 rs5743840 rs352169
rs164640 rs5743848 rs5743839
INDUSTRIAL APPLICATION
[0589] The present invention is directed to methods for assessing a
subject's risk of developing lung cancer. The methods comprise the
analysis of polymorphisms herein shown to be associated with
increased or decreased risk of developing lung cancer, or the
analysis of results obtained from such an analysis. The use of
polymorphisms herein shown to be associated with increased or
decreased risk of developing lung cancer in the assessment of a
subject's risk are also provided, as are nucleotide probes and
primers, kits, and microarrays suitable for such assessment.
Methods of treating subjects having the polymorphisms herein
described are also provided. Methods for screening for compounds
able to modulate the expression of genes associated with the
polymorphisms herein described are also provided.
PUBLICATIONS
[0590] Alberg A J, Samet J M. Epidemiology of lung cancer. Chest
2003, 123, 21s-49s. [0591] Anthonisen N R. Prognosis in COPD:
results from multi-center clinical trials. Am Rev Respir Dis 1989,
140, s95-s99. [0592] Kuller L H, et al. Relation of forced
expiratory volume in one second to lung cancer mortality in the
MRFIT. Am J Epidmiol 1190, 132, 265-274. [0593] Mayne S T, et al.
Previous lung disease and risk of lung cancer among men and women
nonsmokers. Am J Epidemiol 1999, 149, 13-20. [0594] Nomura a, et
al. Prospective study of pulmonary function and lung cancer. Am Rev
Respir Dis 1991, 144, 307-311. [0595] Schwartz AG. Genetic
predisposition to lung cancer. Chest 2004, 125, 86s-89s. [0596]
Skillrud D M, et al. Higher risk of lung cancer in COPD: a
prospective matched controlled study. Ann Int Med 1986, 105,
503-507. [0597] Tockman M S, et al. Airways obstruction and the
risk for lung cancer. Ann Int Med 1987, 106, 512-518. [0598] Wu X,
Zhao H, Suk R, Christiani D C. Genetic susceptibility to
tobacco-related cancer.
[0599] Oncogene 2004, 23, 6500-6523.
[0600] All patents, publications, scientific articles, and other
documents and materials referenced or mentioned herein are
indicative of the levels of skill of those skilled in the art to
which the invention pertains, and each such referenced document and
material is hereby incorporated by reference to the same extent as
if it had been incorporated by reference in its entirety
individually or set forth herein in its entirety. Applicants
reserve the right to physically incorporate into this specification
any and all materials and information from any such patents,
publications, scientific articles, web sites, electronically
available information, and other referenced materials or
documents.
[0601] The specific methods and compositions described herein are
representative of various embodiments or preferred embodiments and
are exemplary only and not intended as limitations on the scope of
the invention. Other objects, aspects, examples and embodiments
will occur to those skilled in the art upon consideration of this
specification, and are encompassed within the spirit of the
invention as defined by the scope of the claims. It will be readily
apparent to one skilled in the art that varying substitutions and
modifications can be made to the invention disclosed herein without
departing from the scope and spirit of the invention. The invention
illustratively described herein suitably can be practiced in the
absence of any element or elements, or limitation or limitations,
which is not specifically disclosed herein as essential. Thus, for
example, in each instance herein, in embodiments or examples of the
present invention, any of the terms "comprising", "consisting
essentially of", and "consisting of" may be replaced with either of
the other two terms in the specification, thus indicating
additional examples, having different scope, of various alternative
embodiments of the invention. Also, the terms "comprising",
"including", containing", etc. are to be read expansively and
without limitation. The methods and processes illustratively
described herein suitably may be practiced in differing orders of
steps, and that they are not necessarily restricted to the orders
of steps indicated herein or in the claims. It is also that as used
herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly
dictates otherwise. Thus, for example, a reference to "a host cell"
includes a plurality (for example, a culture or population) of such
host cells, and so forth. Under no circumstances may the patent be
interpreted to be limited to the specific examples or embodiments
or methods specifically disclosed herein. Under no circumstances
may the patent be interpreted to be limited by any statement made
by any Examiner or any other official or employee of the Patent and
Trademark Office unless such statement is specifically and without
qualification or reservation expressly adopted in a responsive
writing by Applicants.
[0602] The terms and expressions that have been employed are used
as terms of description and not of limitation, and there is no
intent in the use of such terms and expressions to exclude any
equivalent of the features shown and described or portions thereof,
but it is recognized that various modifications are possible within
the scope of the invention as claimed. Thus, it will be understood
that although the present invention has been specifically disclosed
by preferred embodiments and optional features, modification and
variation of the concepts herein disclosed may be resorted to by
those skilled in the art, and that such modifications and
variations are considered to be within the scope of this invention
as defined by the appended claims.
Sequence CWU 1
1
72130DNAArtificialSynthetic 1acgttggatg ctgaattctc ctcagatgac
30230DNAArtificialSynthetic 2acgttggatg aatgcaagtt cttcgtcagc
30330DNAArtificialSynthetic 3acgttggatg aaaactcaga caccaggagc
30430DNAArtificialSynthetic 4acgttggatg agatcaagaa tgagcccgtg
30530DNAArtificialSynthetic 5acgttggatg cctcttattt cagctgctgg
30630DNAArtificialSynthetic 6acgttggatg agagaactct gattctggcg
30730DNAArtificialSynthetic 7acgttggatg accttgcccg tgtggttgaa
30830DNAArtificialSynthetic 8acgttggatg tggcagggta cacagtcaca
30930DNAArtificialSynthetic 9acgttggatg ctgctgtttc tcagagtttc
301030DNAArtificialSynthetic 10acgttggatg gcctgattct tcactacctg
301118DNAArtificialSynthetic 11cctcagatga ctccattt
181219DNAArtificialSynthetic 12cctcagatga ctccattta
191324DNAArtificialSynthetic 13tgttcccctg ggtggacaac tcac
241425DNAArtificialSynthetic 14tgttcccctg ggtggacaac tcacc
251526DNAArtificialSynthetic 15tactcctgcc tctaggaaag accaca
261627DNAArtificialSynthetic 16tactcctgcc tctaggaaag accacac
271716DNAArtificialSynthetic 17ccctgcctgg aggaca
161817DNAArtificialSynthetic 18ccctgcctgg aggacac
171919DNAArtificialSynthetic 19ctgagatgtg ctccttttt
192020DNAArtificialSynthetic 20ctgagatgtg ctcctttttc
202119DNAArtificialSynthetic 21cctcagatga ctccatttt
192225DNAArtificialSynthetic 22tgttcccctg ggtggacaac tcact
252327DNAArtificialSynthetic 23tactcctgcc tctaggaaag accacat
272417DNAArtificialSynthetic 24ccctgcctgg aggacat
172520DNAArtificialSynthetic 25ctgagatgtg ctccttttta
202630DNAArtificialSynthetic 26acgttggatg tgctcaggtg tcattccttc
302729DNAArtificialSynthetic 27acgttggatg ggtggactgg gccatcttc
292830DNAArtificialSynthetic 28acgttggatg ttctgtaacc tggctttctc
302930DNAArtificialSynthetic 29acgttggatg ccaggaattc ccagcttctt
303030DNAArtificialSynthetic 30acgttggatg caaaacaagg gatggcggaa
303130DNAArtificialSynthetic 31acgttggatg aaaggagctg tacctcctcg
303230DNAArtificialSynthetic 32acgttggatg atcagaagag gattcctgcc
303329DNAArtificialSynthetic 33acgttggatg ttcacgcctc cccaggaga
293430DNAArtificialSynthetic 34acgttggatg tatgaactgg gagatgctgg
303530DNAArtificialSynthetic 35acgttggatg tgttgggagt gaggatgtct
303630DNAArtificialSynthetic 36acgttggatg ttgggatgtg ctgttccctc
303730DNAArtificialSynthetic 37acgttggatg agcagagaca taatggaggc
303830DNAArtificialSynthetic 38acgttggatg tgtcaggagg ccttcaggtg
303930DNAArtificialSynthetic 39acgttggatg gttttatgag ggcactggtc
304030DNAArtificialSynthetic 40acgttggatg aggccatagc tgtctggcat
304129DNAArtificialSynthetic 41acgttggatg ttccctttgt ccctggtct
294230DNAArtificialSynthetic 42acgttggatg aggctgcaaa ccagtggaac
304330DNAArtificialSynthetic 43acgttggatg ctgggcaaac aatgaaaatg
304417DNAArtificialSynthetic 44cttccttcct gcagagg
174518DNAArtificialSynthetic 45cttccttcct gcagagga
184624DNAArtificialSynthetic 46ggctttctct tttattttat agtt
244725DNAArtificialSynthetic 47ggctttctct tttattttat agttc
254821DNAArtificialSynthetic 48cccaacccct cctacccgtt c
214922DNAArtificialSynthetic 49cccaacccct cctacccgtt cc
225017DNAArtificialSynthetic 50ggctccttca tcgtccc
175118DNAArtificialSynthetic 51ggctccttca tcgtcccc
185224DNAArtificialSynthetic 52gatgctggta catcccccag gcca
245325DNAArtificialSynthetic 53gatgctggta catcccccag gccac
255417DNAArtificialSynthetic 54gctgttccct ctgcctg
175518DNAArtificialSynthetic 55gctgttccct ctgcctga
185617DNAArtificialSynthetic 56ggagggctcc accctga
175718DNAArtificialSynthetic 57ggagggctcc accctgag
185817DNAArtificialSynthetic 58cctgacctgc tgctgcc
175918DNAArtificialSynthetic 59cctgacctgc tgctgcca
186025DNAArtificialSynthetic 60aacccacaga gctgctttgt atttc
256126DNAArtificialSynthetic 61aacccacaga gctgctttgt atttca
266218DNAArtificialSynthetic 62cttccttcct gcagaggg
186325DNAArtificialSynthetic 63ggctttctct tttattttat agtta
256422DNAArtificialSynthetic 64cccaacccct cctacccgtt ca
226518DNAArtificialSynthetic 65ggctccttca tcgtccca
186625DNAArtificialSynthetic 66gatgctggta catcccccag gccat
256718DNAArtificialSynthetic 67gctgttccct ctgcctgg
186818DNAArtificialSynthetic 68ggagggctcc accctgat
186918DNAArtificialSynthetic 69cctgacctgc tgctgccg
187026DNAArtificialSynthetic 70aacccacaga gctgctttgt atttcg
267125DNAArtificialSynthetic 71ggctttctct tttattttat agttg
257225DNAArtificialSynthetic 72ggctttctct tttattttat agttt 25
* * * * *
References